Basic manufacturing principles are becoming increasingly important in high-throughput sequencing facilities where there is a constant drive to increase quality, increase efficiency, and decrease operating costs. While high-throughput centres report failure rates typically on the order of 10%, the causes of sporadic sequencing failures are seldom analyzed in detail and have not, in the past, been formally reported.Here we report the results of a failure mode analysis of our production sequencing facility based on detailed evaluation of 9,216 ESTs generated from two cDNA libraries. Two categories of failures are described; process-related failures (failures due to equipment or sample handling) and template-related failures .Preventative action based on a detailed understanding of failure modes is likely to improve the performance of other production sequencing pipelines. The average Q20 read length of data generated in the past 12 months (December 2003 to December 2004) from various library types and vector systems is 751 bases. The present study was undertaken to provide insight into the causes of sequencing failures and possible corrective actions. Although our pipeline uses exclusively ABI 3700 and 3730XL automated sequencers, these results should be applicable, in principle, to the improvement of other high throughput sequencing platforms.In the past decade, the demand for DNA sequence data has driven the transformation of sequencing from a research activity into a manufacturing process. High-throughput sequencing facilities are focused on establishing automated procedures that maintain long read length and high overall success rates. It is neither practical nor economical to test each and every DNA template before sequencing . Sequenc20 read length for the entire data set was 771 bases, average pass rate was 87% which was calculated as a percentage of sequencing reactions yielding a minimum of 600 Phred 20 bases. Figure 20 read lengths from the full data set. The analysis methodology employed to determine the failure mode of each trace is outlined in Figure 20 < 600) for further analysis to determine failure mode. The electropherograms from the 1,172 failed reads were evaluated and subsequently categorized into failure mode categories. 64 of these reads were yielded from sequencer capillaries that were clogged and therefore were removed from further analysis and categorized into the "Blocked capillary" failure mode. The remaining 1,108 traces were further classified into nine additional failure mode categories including Low signal strength, Mixed clone with vector sequence, Mixed clone- no vector sequence, Low signal to noise ratio, Excess dye peaks, Hardstop, Repetitive sequence, Homopolymer stretch, and Poly A tail. Results and trace characteristics used to classify each read are as described in Table We generated 9,216 reads from 2,304 clones selected randomly from two cDNA libraries. For each of the two libraries, 1,152 bacterial colonies containing cDNA inserts were picked and arrayed into 384-well microtiter plates  in the preliminary "Low signal strength" category in Table -3, 2.2 × 10-5, and 9.7 × 10-8, respectively. Thus, it is unlikely that more than one read failed per clone by chance.The 164 reads that appear to have sufficient template DNA are grouped by source clone for further analysis. Four possible reads may have failed for each unique clone: 5' replicate #1 (F1), 3' replicate #1 (R1), 5' replicate #2 (F2), and 3' replicate #2 (R2). There were 49 occurrences where two reads failed from one source clone, 2 instances where three reads from the same clone failed and 4 occurrences where all four reads failed from the same source clone. These results are shown in Table Groupings with two failed reads from the same end of the template, "F1/F2" and "R1/R2" showed trace data with noisy background or no signal trace therefore there was insufficient information to determine actual template characteristic leading to the problematic results. With the low probability of seeing two failed traces from one unique clone, we conclude that these failures are most likely a result of template DNA with 5' or 3' ends that are problematic for sequencing. Possibilities include secondary structure, mutated priming sites, long poly A tail, or other homopolymer stretches near the priming sites on the template.The "F1/R1" grouping contained failed reads from both ends of the same replicate but successful end reads from the second replicate. This would indicate that there were no clone related attributes on either end of the template to prevent good sequence thus the failed reads were assessed a process-related failure due to loss of DNA during the precipitation process.The "F1/F2/R1/R2" grouping indicates that no passing sequence data were obtained from 5' or 3' ends of both replicates from the original source clone, all 16 reads showed presence of DNA at each observational check in the process pipeline. The probability that 4 reaction products from a single clone were lost during precipitation is extremely improbable and it is more likely that the original clones had attributes that prevented successful sequencing from either ends. We therefore assign 5' and 3' template-related failure modes for the 16 reads in this grouping.The final groupings of "F1/F2/R1" and "F2/R1/R2" contain 6 reads in total. Each include failed reads from both ends of one replicate and a single failed read from the second replicate. We maintain the same logic for our failure mode evaluation by assessing both a template-related failure to the 2 failed reads from the same replicate and a process-related loss of DNA during precipitation failure for the single read from the second replicate.There were 44 reads in the singleton category where only a single read  failed from each clone. These failures cannot be attributed to the template as three other reads were successfully sequenced from the same source clone. Failure mode was therefore likely to be process related and due to loss of DNA during the precipitation wash steps.20: 500–599 bp bin. This is likely the result of a presence in greater amount of one reaction product over the second leading to proportionately stronger signal strength.Having established a stringent pass criteria of 600 Phred 20 bases or greater, we observed a broad range of failure modes distributed among the failed reads. Reads less than 100 bp did not yield useable data and were usually a result of process-related failure modes. Process-related mishaps in the pipeline would include missed colony picks, blocked capillaries on sequencers, faulty tips on automated liquid handling devices, or lost DNA template during precipitation. The sequences generated have low average read lengths, or in cases where DNA is totally lost, zero read length. 88% of reads within the largest "process" related failure mode category, "low signal, no DNA", were a result of failed cultures at the beginning of the process pipeline. The absence of DNA was verified early on by failed growth in the source microtiter plates and further confirmed by failed growth in the stamped replicate agar plate. Failures are perhaps due to missed bacterial colony picks and adjustments such as recalibration or refining the morphology criteria of the colony picker should help. The second most prevalent failure mode in the "process related" category, "Mixed clone with vector sequence" (30% of reads in this classification), is likely a result of two side by side colonies being picked into the same well or through contamination originating from the use of automated liquid handlers. These problems might be alleviated by increasing the stringency of the proximity criteria on the colony picking device to ensure sufficient distance exists between two bacterial colonies before they are chosen for picking. As we use new tips on the instruments daily, increasing the length or number of wash cycles on the automated liquid handlers between volume transfers may help in reducing carry over contamination from one plate to another. This will need further investigation. Cross contamination between adjacent wells can also arise with the mishandling of plates, or when tips are plunged too deeply into the wells, displacing the volume into adjacent wells. Adjusting the liquid handler by reducing the depth and speed of tips entering each well may help prevent the occurrences of cross contamination within a plate. Despite the presence of mixed clones within one well, traces often yielded discernable sequence with read lengths broadly distributed up to the Q23N  anchored primer to resolve the 3' ends of cDNA inserts.Template-related failures often still yielded reads of several hundred base pairs. Template characteristics that might contribute to failed or truncated reads could be poly A tails, homopolymer stretches, or other highly repetitive regions. Problems in sequencing these types of regions are well cited in the literature and are dependent on chemistry and methodology used in sequencing. These problems are best avoided by using an anchored primer for first strand synthesis of cDNA as the first step in library construction. "Poly A tail" was the single largest cause of failed sequence in both the template-related failure category as well as the entire data set, yet the reads make up the majority in the higher failed read length bins. In our regular production pipeline, we often circumvent this problem by using a combination of a 3' primer to resolve sequence immediately following the poly A coupled with an oligo(dT)The FMA pipeline described here was tailored to our high-throughput 384-well automated sequencing pipeline but many of the components in this platform are shared within the high-throughput sequencing community such as the alkaline lysis procedure to prepare and purify DNA, DNA sequencing using Big Dye chemistry, ethanol precipitation to clean up reaction products and equipment such as the Genetix QPIX, Beckman Coulter Biomek FX, and Applied Biosystems 3730 xl DNA analyzer. For this reason our FMA methods can be readily adapted to analyze other similar sequencing platforms. Extending our present study to other library types commonly sequenced in the high-throughput community, such as shotgun or Serial Analysis of Gene Expression (SAGE) should offer further information regarding template specific failures. This failure mode analysis provides information on distinguishing between process and template-related attributes that may lead to downstream failed sequence. It can therefore be a useful tool used to audit overall sequencing procedures and identify key problematic steps in a process pipeline. Making proper adjustments to the pipeline based on the results will likely result in increased efficiency, enhanced data quality, and decreased cost.Samples are processed in duplicate, with quality assurance and observational checks to account for status of each read after every procedure leading to the final sequence read. These recorded observations facilitate downstream systematic analysis of failed sequence data. The regular core production pipeline was not altered for this study, as the purpose is to assess sequence failure modes under ordinary conditions in a high-throughput sequencing environment. An overview of the observational checkpoints within the FMA pipeline is outlined in Figure Populus trichocarpa cDNA libraries were transformed by electroporation into 40 μl of E. coli DH10B T1 resistant cells (Invitrogen). Transformed cells were recovered using 1 mL of SOC medium (prepared in house) and plated onto 22 cm × 22 cm agar plates (Genetix) containing 2xYT agar and 100 μg/μl Ampicillin. Agar plates were incubated overnight at 37°C for 14 hours. Bacterial colonies were picked from the agar plates and arrayed into 384-well microtiter plates (Genetix) containing 60 μl of 2xYT medium + 7.5% glycerol (made in house) using the Genetix QPIX automated colony picker (Genetix). A total of six 384-well microtiter plates, three plates for each of the two cDNA libraries, were picked. The plates were incubated overnight at 37°C for 16 hours then each microtiter plate was inspected for wells that contained no growth. The positions of all failed cultures were recorded. To further verify growth in each well after the incubation period, a disposable 384-well replicator was used to stamp bacterial culture from each 384-well microtiter plate onto a new 22 cm × 22 cm agar plate containing 2xYT agar and 100 μg/ul ampicillin. Agar plates were incubated overnight at 37°C for 14 hours then inspected the next day for colonies from every well. The positions of all failed growths were recorded.One microliter of ligation mix from each of two Two microliters of bacterial culture was transferred from the 384-well microtiter plate into a 240 μl 384-well deep well diamond plate (Axygen) containing 60 μl of 2xYT medium and 100 μg/ml ampicillin using a 384-well slotted inoculator (V&P Scientific). This was done in duplicate to create two sets of six 384-well deep well inoculated diamond plates. Inoculated plates were sealed with AirPore™ tape (Qiagen) and placed into a 37°C shaking incubator (New Brunswick Scientific C25 Incubator Shaker) at 350 rpm for 18 hours. After the incubation period, cultures were removed and each plate inspected for growth and contamination. All failed cultures were recorded. Cultures from both replicates were then placed onto separate multi-tube floor vortexers (VWR) at maximum speed for approximately 5 minutes until all cells were resuspended. Cultures were stored at 4°C until ready for DNA preparation. DNA was prepared using alkaline lysis [DNA preparations were evaluated by agarose gel electrophoresis. A 1.5 μl aliquot of purified DNA was combined with 1.5 μl of bromophenol blue loading buffer  and 2 μl was loaded onto a 1.2% agarose gel. Samples were loaded using a 12-channel loader (Hamilton) beside 3 ng of 1 kb plus DNA marker (Invitrogen). Gels were run at 120 volts for 90 minutes in TAE (Tris/Acetate/EDTA) buffer followed by staining for 35 minutes in SybrGreen Nucleic Acid stain (Cambrex). Gels were scanned using a Fluorimager 595 (Molecular Dyanmics) scanner. The image was visually examined for genomic DNA, as well as presence and quality of DNA. All empty lanes and observations were recorded and entered into the FMA database.DNA Sequencing reactions were assembled in 384-well clear optical reaction plates (Applied Biosystems) using a Biomek FX workstation (Beckman-Coulter). In each 5 μl reaction  the following were added: 3 μl of purified plasmid DNA (~45 ng/μl), 0.26 μl of sequencing primer , 0.43 μl of 5X reaction buffer (Applied Biosystems Big Dye Terminator 5X Sequencing Buffer), 0.77 μl of Ultrapure water (Gibco), and 0.54 μl of BigDye v.3.1 ready reaction mix (Applied Biosystems). Each well of the reaction plate was visually inspected for appropriate volumes after both reaction mix and DNA addition. Volumes were manually adjusted using a single channel pipet (Gilson) where required and all observations were recorded. Sequence data were obtained using universal M13 Forward (5'-TGTAAAACGACGGCCAGT-3') and M13 Reverse (5'-CAGGAAACAGCTATGAC-3') primers on each set of replicate plates. Thermal cycling was performed on PTC-225 thermal cyclers (MJ Research) with parameters of 35 cycles at 96°C for 10 seconds, 52°C for 5 seconds using M13 Forward primer or 43°C for 5 seconds using M13 Reverse, 60°C for 3 minutes, followed by incubation at 4°C. Reaction products were precipitated by adding 2 μl of 125 mM EDTA (pH8) and 18 μl of 95% ethanol per well followed by centrifugationat 2750 × g for 30 minutes in an Eppendorf 5810R centrifuge. The EDTA/ethanol was immediately decanted and reaction products washed with 70% ethanol. The 384-well cycle plates were allowed to dry inverted for 15 minutes. Samples were resuspended in 10 μl of Ultrapure water and analyzed using a 3730XL DNA analyzer (Applied Biosystems). The performance of each capillary on the four DNA analyzers used in this experiment were validated using one 384-well control plate for each instrument. Each 384-well plate contained our in-house control standard, a full-length human cDNA clone obtained from the I.M.A.G.E. Consortium . Blocked capillaries were recorded into the FMA database and traces originating from these capillaries flagged. Sequence data were evaluated using PHRED software [GSY was responsible for design and execution of this study, plus analysis of data and drafting of the manuscript. RAH conceived of the study and directed the analysis. JMS and SAB generated the data described in the study and participated in study design. DS developed several of the key protocols used in this study. MB provided quality assurance of the experimental procedures and participated in study design. MAM participated in the establishment of the production pipeline and read the manuscript and provided comments.


The practice of continuous, long-term monitoring of human joint motion is one that finds many applications, especially in the medical and rehabilitation fields. There is a lack of acceptable devices available to perform such measurements in the field in a reliable and non-intrusive way over a long period of time. The purpose of this study was therefore to develop such a wearable joint monitoring sensor capable of continuous, day-to-day monitoring.A novel technique of incorporating conductive fibers into flexible, skin-tight fabrics surrounding a joint is developed. Resistance changes across these conductive fibers are measured, and directly related to specific single or multi-axis joint angles through the use of a non-linear predictor after an initial, one-time calibration. Because these sensors are intended for multiple uses, an automated registration algorithm has been devised using a sensitivity template matched to an array of sensors spanning the joints of interest. In this way, a sensor array can be taken off and put back on an individual for multiple uses, with the sensors automatically calibrating themselves each time.The wearable sensors designed are comfortable, and acceptable for long-term wear in everyday settings. Results have shown the feasibility of this type of sensor, with accurate measurements of joint motion for both a single-axis knee joint and a double axis hip joint when compared to a standard goniometer used to measure joint angles. Self-registration of the sensors was found to be possible with only a few simple motions by the patient.After preliminary experiments involving a pants sensing garment for lower body monitoring, it has been seen that this methodology is effective for monitoring joint motion of the hip and knee. This design therefore produces a robust, comfortable, truly wearable joint monitoring device. Long-term measurement of human movement in the field is an important need today . For manMany specific medical applications benefit from the information provided by continuous human movement monitoring. To better develop and optimize total joint replacements, for instance, a detailed record of a patient's daily activities after such a replacement is required . The meaPresently, there is no satisfactory solution for long-term, human movement monitoring in the field. The use of video and optical motion analysis systems offer the most precise evaluation of human motion, but obviously restrict measurements to a finite volume . Body moElectrogoniometers are frequently used to measure dynamic, multi-axis joint angle changes in individuals, providing continuous joint movement information. These devices, however, are not desirable for long-term monitoring of daily living, since they are exoskeletal devices that cross the joint, potentially interfering with movement. Furthermore, any shift from their original placement leads to errors in angle estimations . Such coOther types of goniometric devices have been developed for measuring particular parts of the body. Electronic gloves -13, for For all types of body-mounted sensors, the issues of comfort and wearability are of major importance, if a patient has to wear the monitoring device for extended periods of time. Furthermore, such home-use wearable sensors need to be put on and off every day without close supervision of a medical professional. Proper registration of the sensor is therefore a crucial requirement for deploying wearable sensors to the home environment.The goal of this paper is therefore to develop a new method for continuous monitoring of human movement by measuring single or multi-axis joint angles with a wearable sensing garment that is non-intrusive and non-cumbersome and that can be properly registered for reliable monitoring. A new method is presented here for joint monitoring using conductive fibers incorporated into comfortable, flexible fabrics. All that is needed is a one-time calibration with a standard goniometer, and a conductive fiber sensor garment is then able to continuously detect joint movement and measure specific single or multi-axis joint angles. With an array of sensors incorporated into a sensing garment, registration of the sensor occurs automatically each time the garment is worn through only a few simple motions by the wearer. This type of wearable sensor would allow extended home monitoring of a patient, and is no harder to put on than a typical article of clothing.In the following, the principle and design details of this wearable device will be presented, along with effective algorithms for allowing a patient to perform long-term, unsupervised monitoring in the home environment. Experimental feasibility tests will also be presented on a prototype wearable sensor for both single-axis and multi-axis joints.The basic principle behind the wearable sensors presented in this paper is as follows: when a particular joint on the human body moves, skin around the joint stretches, along with any clothing surrounding the joint as well. A former study by the textile industry has shown that body movements about joints require specific amounts of skin extension. Lengthwise across the knee for example, the skin stretches anywhere from 35–45% during normal joint movement .When a particular joint moves, fabric around the joint will either expand or contract accordingly, assuming the fabric is form fitting to the skin, and has the necessary elastic properties. To assure comfort and freedom of body movement, stretchability of 25 to 30 percent is recommended for fabrics fitting closely to the body . By incoA in the figure. Along the conductive fiber, there is a wire contact point at B that is permanently stitched into the fabric. The other end of the conductive fiber, point C, is kept in tension by a coupled elastic cord, which is permanently attached to the remote side of the joint, point D. Therefore, any stretching in this coupled material will take place in the highly elastic cord, CD, and not in the conductive fiber AC. As the joint moves, the elastic cord will change length, causing the coupled conductive fiber to freely slide past the wire contact point at B that is stationary. The conductive fiber always keeps an electrical contact with this wire, but the length of conductive thread between points A and B will change as the joint rotates. The resistance, which is linearly related to length, is then measured continuously across these two points A and B.Figure θ1, θ2, and θ3. Our goal is to estimate these joint angles based upon the output of sensors 1, 2, and 3.Consider Figure Preliminary experiments have shown a clear relationship between joint angle and sensor output for individual sensors about various joints of the body. Figure It is desired to design a filter that receives sensor signals as inputs, and predicts the joint angle(s) of interest. In the proposed method, each joint angle being monitored has a corresponding single sensor that is situated about that particular joint for maximum sensitivity, as in Figure N axis sensors for measuring N joints, each consisting of a single thread sensor, as shown in Figure Consider  is the N × 1 vector of N joint angle predictions,  is its bias term, y = (y1 … yN)T is the N × 1 vector of corresponding sensor readings, and G and  are, respectively, the N × N matrix and the N × 1 vector experimentally determined to relate the inputs and the outputs.where Since there is a slight amount of curvature in the preliminary data of Figure whereG' is an N × N(N-1)/2 experimentally determined matrix. The three terms on the right hand side of the above equation can be incorporated into a homogeneous expression using augmented matrix and vector:and W and Y arewhere W = (θ0 G G')     (5)W, a least squares regression is performed using m sets of experimental data from a collection of sensors on an individual patient. Let P be a N × m matrix consisting of m sets of experimentally measured joint angles,To determine the parameter matrix B be a {1 + N(N + 1)/2} × m matrix containing the corresponding sensor outputs and their quadratic terms:and B = (Y(1) … Ym)     (8)W* that minimizes the squared prediction errors is given byThe optimal regression coefficient matrix W* = PBT (BBT)-1     (9)BBT non-singular.if the data are rich enough to make the matrix product N axis sensors. In practice, however, they can be reduced to a compact expression with lower orders. First the offset  can be eliminated from the coefficient matrix W, if the sensor outputs are zeroed at a particular posture, e.g. the one where the extremities are fully extended. Second, although the matrix G contains off-diagonal elements representing cross couplings among multiple joints, some joints have no cross coupling with other joints. For example, the measurement of the knee joint can be performed separately from that of the hip joints. If the j-th joint is decoupled from all others, it can be treated separately as:The above expressions are the most general forms for G, their second-order cross coupling terms, e.g. yj yk, can be negligibly small with proper design of individual sensors. In such a case, two coupled joints, say j and k, can be written as:where the offset is eliminated. Third, although multiple joints are coupled to each other having non-zero, first-order off-diagonal coefficients in matrix where the offset terms have been eliminated. Thus the number of parameters to identify through calibration experiments is reduced. In consequence, the dimension of the optimal coefficient matrix must be reduced accordingly. The same calibration procedure is performed for both single axis and multiple axis cases, and need be performed only once for a specific set of sensors on an individual.Although one sensor is sufficient to capture single-axis joint motion, any misalignment of such a sensor from use to use will lead to erroneous measurements. From a practical standpoint, it is obvious that a method is needed to adjust for any shifting of a sensor about the joint that will take place from one use to the next. It is both undesirable and impractical to recalibrate the whole sensor every time the patient takes off the sensing garment and places it back again. To take care of such registration problems, an array of multiple sensor threads is used. By incorporating multiple threads in a known pattern, a template-matching algorithm can be performed to determine a sensor's offset from calibration. In this way, measurement errors due to sensor misalignment are significantly reduced. The details of this method are described in the next section.The goal in designing these wearable sensors is to create a device that is ultimately self-registering for subsequent uses after the initial one-time calibration experiments. This means that no additional equipment is needed to register the sensors for each use. Also, it is important that any procedures that are needed for self-registration are simple, and able to be preformed by the patient without supervision. To achieve these goals, a multi-thread sensor array design is presented.M sensors covering a single-axis joint as shown in Figure d. This multi-thread sensor array is used to estimate a single-axis joint angle, θj. To develop a registration procedure let us first calibrate each sensor thread individually. Let  be the estimate of the j-th joint based on the i-th thread sensor given byFirst, consider an array of where is the 1 × 2 regression vector that is optimized for the i-th single-thread sensor of the j-th joint placed at a home position.and α, from the original position where calibration was performed. See Figure α. Assuming that the individual sensor threads are identical other than being separated by a distance d, we can conclude that the pattern of the sensitivity array is a shifted version of the calibrated one, as shown in the simplified plots of Figure Now consider the situation where the sensor array has been removed, and placed back on the joint for more measurements. The sensor array is now offset an unknown distance,  will no longer be the appropriate regression matrix to estimate θj from Yj(i). A new, unknown vector  will instead relate the sensor output to θj: is unknown, each individual sensor in the array should ideally give the same estimate for the actual joint angle at any time, so thatAlthough If the shifting of the sensor array were to happen in a discrete fashion,α = nd     (16)n is an integer value, it is seen thatwhere n is an unknown, it is desired to find an n that satisfies (15) and (17), rewritten asSince n is assumed to be |n| <M - 1. Namely, the sensor array, although shifted, can still cover the joint, having an overlap with the original sensor at the home position.where n that can be found to exactly solve (18). Unfortunately, for practical usage, n will not be a discrete integer. Furthermore, n cannot be explicitly found since process and measurement noise will cause the sensor outputs to deviate from their "ideal" values. With the knowledge of  for i = 1 ~ M, however, it is possible to find the optimal integer n that best solves (18).In the ideal, theoretical case, there will exist an integer n as follows :Let us first define the average joint angle estimate for M threads of sensor outputs for a given integer n is found by minimizing the average squared error between each sensor's estimate and the average estimate with respect to n (i.e. reducing the variance in the estimated angle as a function of n):The best estimate for n = -M+2, -M+3, ..., M-3, M-2. The value for  found from (20c) is then used in (17) to approximate each sensor's predictor regression matrix for this new offset position of the array. In the ideal discrete case, where α = nod, no is the discrete offset of the sensor array,  = no, and Rj = 0.Equations (20a) and (20b) are solved for a is not a discrete multiple of d, the minimum variance is not zero, Rj  ≠ 0, but it will decrease as M increases, and d decreases.For the non-ideal case, where θj. Furthermore, since  can always be approximated using this algorithm, a one-time calibration is all that is needed for these wearable sensors to be used by a patient.Creating a denser sensor array in this way leads to more accurate estimates of sensor sensitivities, which in turn leads to more accurate estimates of  is converged upon. Again, the denser the array of sensors used, the better the estimate obtained. Following this, the algorithm need not be performed as often, as long as the sensor array remains stationary for an individual use. To begin monitoring, it is assumed that  = 0.The registration algorithm takes place in real time as the sensor is in use. All that is needed for a patient to begin using these sensors is to first "zero" the sensor output with the joint fully extended in the 0° position, and then freely move the joint to obtain non-zero data. This non-zero data will then allow the self-registration to take place. While registration is not needed at all times, it should be performed during initial operation until an appropriate d. The j-th joint array is placed so that it is most sensitive to changes in θj, while the k-th joint array is situated so that it is most sensitive to changes in θk.In the double axis case, two sensor arrays are placed around a predominantly two-axis joint such as the hip. As in the single-axis case, each array contains M sensor threads equally spaced by a distance θ1 alone. This hip flexion/extension causes significant changes to sensor array 1, y1(i), i = 1 ~ M. Next the patient is instructed to make hip abduction/adduction (θ2) alone, which causes significant changes to sensor array 2, as shown in Figure θ1 or θ2, has been moved, since sensor array 1 is most sensitive to θ1, and sensor array 2 for θ2. Once the individual axis movements are detected, the same registration procedure as that of a single axis can be applied to determine the misalignment of each sensor array. Once the misalignment is determined, the corrected, optimal predictor can now be used for verifying whether the registration has been performed correctly based on individual axis movements.For registration, let the patient move only one axis at a time. As illustrated in Figure This registration method reduces the multi-axis problem to individual single axis procedures. However, the single axis procedures do not have to be repeated for all axes, if they are tightly related. For the two hip axes in Figure All experiments have been conducted under a protocol approved by the Massachusetts Institute of Technology Committee on the Use of Humans as Experimental Subjects .Figure In these particular pants, an array of eleven sensors spans across the knee joint, each separated by a distance of 5 mm, and each with an unstretched length of 55 cm. The sensors threads were silver plated nylon 66 yarn, which had an impedance of approximately 3.6 Ω/cm. Single sensors span both the posterior and side of the hip as well to capture two axes of hip motion. These single sensors are not seen in the view of Figure To get an idea of the capabilities of existing technology available for joint monitoring, tests were initially performed using a standard electrogoniometer. Figure The goniometer was taken off and placed back on the knee joint eight separate times. Each time the goniometer was put on, the leg was extended (Position 1) and the goniometer output was set to 0°. The leg was then slowly bent to Position 2 (50°) and the goniometer output was recorded. The average rms error between the goniometer output, and the known joint angle (50°) for these tests was 3.5° with a standard deviation of 2.6°. Even with the goniometer placed on the same joint by the same person, these results illustrate the fact that slight changes in how the goniometer is attached can lead to varying measurements. It will be important to keep errors such as these in mind when the results from the conductive fiber sensor are analyzed.Having just discussed the possible errors introduced by a standard electrogoniometer, it is important to also highlight the possible errors introduced by a conductive fiber thread sensor. Consider again Figure As can be seen, there is a significant amount of variation possible in sensor output for a given joint angle. In particular, for threads over the knee joint, the average rms error between curves such as those shown in Figure The pants sensing garment was first used to estimate single-axis knee angle measurements. For the following single-axis experiments, a rotary potentiometer firmly attached to the leg was used as a goniometer, and this was the standard for which to compare joint angles. In each experiment, the potentiometer was "zeroed" with the leg in the full extension position.A calibration was performed to find the optimal regression matrix for both the linear and nonlinear predictors, and a sequence of knee movements was then monitored with the sensors. Figure The performance of the pants sensors can be seen to be quite good, accurately capturing the joint movement patterns over time. The average rms error between the pants sensor estimate and the potentiometer using the linear predictor was 5.4°, while that for the quadratic predictor was significantly better, at just 3.2°.It is important that these sensors are able to measure all types of motion, including higher frequency motion. To determine the frequency capabilities of the prototype fiber sensors, tests were performed where the leg was swung back and forth at different frequencies. The resulting sensor estimations, and errors when compared to the potentiometer, are summarized in Figure From these results, it is seen that the sensors are able to track the joint motion for frequencies as high as 2 Hz, but significantly larger errors result as the frequency is increased. Since most gross human motion takes place below these frequencies in a typical day, these sensors are suitable for everyday measurements, but such limitations should be considered if more accurate measurements are desired.Since these sensors are to be worn multiple times by a user, the reliability of registration is important every time the sensors are worn. Therefore, it is important that using the template-matching algorithm with an array of sensors will give an accurate registration each time the sensors are taken off and put back on. To verify this, an initial repeatability test was performed on the prototype sensor pants. The pants sensors were taken off and put back on four separate times to simulate four future uses of the sensors after an initial calibration test. The knee joint was moved over a wide range of motion in each instance. The joint angles measured by the fiber sensors for each test are shown in 11. The errors between these measurements and the potentiometer measurements are summarized in Table Again, the sensors are able to capture the overall motion of the knee in each case, but appear to give less accurate results each time the pants are worn. For this reason, while a completely self-calibrating sensor is always desirable, it may be necessary to re-calibrate the sensors after many uses for more accurate measurements.y1 and y2, spanning the posterior and lateral side of the hip, respectively . In the second segment, leg movement was allowed only in the frontal plane, while the subject performed an abduction/adduction movement three times .Figure In each case, the sensor spanning the axis in which the angle changes took place was the most sensitive to change, as expected. Each joint motion also produced small, but not insignificant, cross-coupling outputs in the "remote" sensors as well, showing that a single sensor output is dependent on multiple joint angles, and not one single angle.The pants sensor threads about the hip joint were then calibrated with the twin-axis goniometer. Table After initial calibration, random leg movements were then monitored with the sensors. Figure The pants sensors were again able to capture the joint movement patterns over time, in this case for two axes of motion. The average rms error between the pants sensors' estimate of hip flexion angle and the goniometer's was 2.5° using the linear predictor and 2.4° using the quadratic predictor. For hip abduction, these errors were 2.1° and 1.7° respectively. In this double axis case, the differences between the linear and quadratic predictors were not very significant over the typical ranges of hip joint angles measured.Previously, the assumption was made for the double axis hip joint that both sensor arrays would be offset from their calibration position by the same amount for each use. This allowed the double axis registration to be reduced to a single axis registration. To verify this assumption, a simple experiment was performed on the pant's hip sensors. The pants were taken off and put back on ten times. Each time, the distance around the waist between the sensor thread on the side of the hip, and the sensor thread on the rear of the hip was measured  movement of the fiber across the wire contact point may affect sensor output due to uncertainty in the area being contacted, and dynamic effects of the constant rubbing action; 2) although the elastic cord takes up a majority of the sensor tension, slight changes will also take place in the fiber tension as the joint is moved, and this will affect fiber resistance; and 3) different sections of even the same fibers will exhibit slightly different resistance characteristics due to the slightly inhomogeneous nature of such fibers. In spite of all these sources of uncertainty, it is still possible to accurately calibrate a set of sensors, and achieve acceptable joint measurements with minimal errors. These effects are minimized through careful selection of the particular fibers used as sensors, and in manufacturing the garment.While two specific predictor models have been presented for the calibration of a set of sensors, there are of course many more candidates that could be used as well. The linear and quadratic models used in this paper were the simplest choices, and the experimental results showed no advantage to adding more terms. Doing so only increased the computational requirements unnecessarily. This is why the models were presented as they were.A few more words should also be said about the registration algorithm. As presented, this algorithm only accounts for shifting of a set of sensors in one direction . It is felt that this is appropriate due to the construction of the sensing garment. With the sensors instrumented in a "vertical" fashion, the user is responsible for visually checking that they put the garment on with no twist. This is relatively easy to do with the fibers oriented vertically. Furthermore, as long as the sensors span well beyond the local effects of skin movement around a joint, small shifts in the vertical direction will theoretically have little to no effect on the sensor output. Requiring a patient to "zero" the sensor output with all joints in the 0° position each time the garment is worn further eliminates any errors due to sensor drift.Finally, the wearability of the pants sensing garment must be addressed. What makes this sensing garment "more wearable" than existing joint measurement devices is that it is simply a pair of pants that people already wear on a regular basis.The extra sensors and wires added to these pants are compact and lightweight, almost negligible to the wearer. These sensors are easy to use, requiring much less skill and carefulness by the user, in general, than a typical goniometer.A wearable joint movement sensor design has been presented that uses conductive fibers incorporated into a fabric that is form fitting to a joint. Resistance changes in the fibers caused by fiber movement as the joint is moved can be related to angular joint position. Using multiple fiber sensors, multi-axis joint angles can be determined, in addition to single-axis angles, after a one-time calibration procedure performed by a therapist/physician. Implementing a nonlinear predictor model, continuous joint angle measurements can be made during daily activities, with the sensor able to be taken off and put back on at any time with no need for manual recalibration. Sensor offsets due to misregistration can be accounted for through the use of a sensor array spanning the joints of interest. This allows the sensors to self-calibrate, with only a few simple motions of the patient.After preliminary experiments involving a pants sensing garment for lower body monitoring, it has been seen that this methodology is feasible for monitoring joint motion of the hip and knee. Multiple sensor arrays are used at multi-d.o.f. joints, where each sensor output is coupled to multiple joint angle changes. This design therefore produces a robust, comfortable, truly wearable joint monitoring device. This paper outlines the development of this sensor from initial idea to working prototype. Future effort is needed in developing a completely wearable, highly accurate sensor, though. This would include making the sensors wireless, and therefore "tether-free." More precise textile manufacturing techniques would also be needed to further reduce measurement errors.The author(s) declare that they have no competing interests.PTG developed the ideas discussed in this paper under the guidance of HHA. PTG carried out all experiments. Both authors read and approved the final manuscript.


The management of burn injuries is reported as painful, distressing and a cause of anxiety in children and their parents. Child's and parents' pain and anxiety, often contributes to extended time required for burns management procedures, in particular the process of changing dressings. The traditional method of pharmacologic analgesia is often insufficient to cover the burnt child's pain, and it can have deleterious side effects ,2. InterThis study investigates whether playing a virtual reality game, decreases procedural pain in children aged 5–18 years with acute burn injuries. The paper reports on the findings of a pilot study, a randomised trial, in which seven children acted as their own controls though a series of 11 trials. Outcomes were pain measured using the self-report Faces Scale and findings of interviews with parent/carer and nurses.The average pain scores  for pharmacological analgesia only was, 4.1 (SD 2.9), while VR coupled with pharmacological analgesia, the average pain score was 1.3 (SD 1.8)The study provides strong evidence supporting VR based games in providing analgesia with minimal side effects and little impact on the physical hospital environment, as well as its reusability and versatility, suggesting another option in the management of children's acute pain. Pain is a noxious stimulus which can be interpreted in many ways by different individuals but as yet the mechanisms by which the body manages it are not completely understood. Pain has been defined by the International Association for the Study of Pain as 'an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage' . Though Children with burn injuries undergo significant physical and emotional trauma, initially from their injury, and subsequently from the dressing changes and related treatment they undergo throughout the healing phase. The latter is referred to, in this article, as 'procedural pain'. Clinicians involved in the care of children with acute burns use the best available methods to reduce procedural pain. Adequate and appropriate pain management is essential to ensure that symptoms secondary to pain experiences do not become habitual . Moreoveanalgesics, muscle relaxants and hypnotics. These drugs help in reducing procedural pain experienced by children, however they frequently have unwanted side effects such as drowsiness, nausea, reduced postural control and lethargy [The burns service at the Women's and Children's Hospital  collaborated with University of South Australia, on this project. Pain management comprises administration of a number of medications, including lethargy ,8.Pain management of children in hospitals during dressing changes has been reported as inadequate ,2 and isVirtual Reality (VR) was initially conceived as a tool for pain modulation by Hoffman et al -18 who fWhy virtual reality? VR can be considered intermediary to reality and computer technology. Owing to its ability to allow the user to immerse and interact with the artificial environment that he/she can visualize, the game-playing experience is very engrossing -18,20-22This project was an interdisciplinary and inter-sectorial  collaboration between the Centre of Allied Health Evidence, and the  Department of Computer and Information Sciences, both at the University  of South Australia (UniSA), Australia, and Women's and Children's  Hospital(WCH), Adelaide, South Australia.Ethical approval for this project was obtained from both the institutions .All children admitted to one specific ward (Newlands Ward), WCH, aged between 5 and 18 years, having burns to more than three percent of their body surface area, and requiring dressing changes, were eligible for inclusion in the study. Children with burns to their hands, face or head, past history of epilepsy and reduced intellectual capacity were not included, as they would have been unable to appropriately use the VR equipment.All eligible subjects were identified by ward staff, and were invited to participate in the project by the project team. Written child and parent consent was obtained at every contact.The test administrations of routine pharmacological analgesia or routine pharmacological analgesia coupled with virtual reality were randomly assigned to each half of the burns dressing change  following a coin toss determining the sequence. The child and parents were given a standard explanation about the VR administration and the VR game. If required, subjects were allowed a short preview to assist them to understand how to play the game.• The VR equipment constituted a laptop  with the game software, developed by the Department of Computer and Information Sciences, UniSA (Based on the game 'Quake' by ID Software), a head-mount display (HMD) (IOGlasses Head Mount Display with a SVGA video resolution of 800 × 600 16 million colours), with a tracking system , to allow interaction with the virtual environment by moving the head and neck and decoder and a mouse used as a trigger. The game involved a visual simulation giving the children a feel of being on a track, using a pointer to aim and shoot monsters. Figure • The developers considered the applicability of the game through varying age groups, gender, intelligence and intellectual capacities, while designing the game. The game tried to achieve effective distraction via immersion without violence and a simplified game structure requiring minimal control by the player, to allow the smallest possible movement during the dressing change procedure.A within-subject design was used in which the children acted as their own controls.There was no interference with the dosage or type of analgesia which was administered to the children 30 to 45 minutes prior to dressing removal.The burns dressing changes would normally occur every Wednesday morning before the hospital ward, grand rounds. The dressing change involved administration of prescribed medication and application of olive oil on the dressing (if adhesive tape was covering the wound), approximately 30 to 45 minutes prior to the actual procedure. The first treatment half constituted the removal of the adhesive tape/bandages and the under-dressing (acticoat/silver oxide dressing) and the second half comprised of the wound being debrided and a fresh dressing applied, after being assessed by the consultant medical officer/s.Following the completion of each half of the dressing change (with or without administration of VR), the researcher obtained scores for average pain using the Face Scale Figure , and intThe subjects were asked to score their average pain experience at the end of each phase of the dressing change procedure . Pain was scored using a modified self-report Faces pain scale . The scaThe data was analysed by a blinded assessor, to reduce any biases and increase the rigour with which a de-identified and coded dataset was probed.There were nine eligible, consenting child subjects (6 boys and 3 girls) in the sample. The average age for both boys and girls was 10.0 years (SD 3.7 and 4.1 respectively), age ranging between 5 to 16 years. The average percent of body surface area burnt was 5.3% (SD 3.4%) and there was no significant gender or age difference in body area burnt.For boys, two had burns from contact with a silencer (muffler) on a four wheeled motor bike, two were burnt from a hot water bag bursting, one was burnt while playing with petrol and fire, and the remaining boy was burnt with hot oil from a BBQ. For the girls, all three were burnt by overturned fluids. All participants were experiencing burns for the first time, and when enrolled into the study, their burns were at either second or third dressing change. Every participants' pain, prior to enrolling in this study, had been managed either with no pain relief, or by pharmacological means.For every child participant, one parent or guardian with one exception (was not available to observe the dressing change) provided data on the effectiveness of the VR for every post-session interview. One key nurse involved in the burns management was also interviewed following each session.Overall, 13 trials were undertaken from nine children . The results of two subjects were withdrawn for further analysis as the respective participants were too drowsy from the effects of analgesia to participate appropriately in the VR section of the session. Thus the remaining seven child subjects were included for analysis, with a total of 11 useable trials . The seven participants in the included trials had an average age of 11.1 years (SD 3.5).p <0.05) in time taken in the two treatment halves . The average difference in administering the two treatment halves was approximately 2 minutes , over all included trials was 4.1 (SD 2.9), whilst for VR coupled with pharmacological analgesia, the average pain score was 1.3 (SD 1.8). Because of the small number of child subjects in the study, the data was considered per child, and per trial. Over all included trials, the mean pain score difference between administrations was 3.2 (SD 2.1), which was significant using paired t-tests (p < 0.01). This indicated the importance of the effect of using VR  in reducing pain experiences during burns dressing changes. The per trial pain responses to VR and analgesia, and analgesia alone, compared with the average trial response per administration is shown in Figure For each child subject who completed an eligible trial, the average per-child difference in pain scores between administrations of VR & Pharmacological Analgesia, or Pharmacological Analgesia alone, suggested that every child but one obtained an improvement in pain scoring of at least 2 points on the Faces Scale, attributable to VR, as demonstrated in Figure All nurses or parents agreed that VR helped distract the children  and was helpful in reducing pain and there were no negative comments  regarding the application of VR. (note - delete 'interestingly')The overwhelming response from the nursing staff was that VR administration was helpful to the child. Comments from the interviews are provided below as evidence of this."... probably VR helped to take concentration off ... probably helped take away a lot of the anticipation away from the treatment"."... from my past experience, I can tell that it (changing burns dressing) can be a real problem. It was not a problem today. He did not even flinch while the dressing was being taken off.""... communication was good – he understood what I asked him to do. I did not find it invasive or intrusive...""... it was great to do the changing (of dressing) without dumping him with medication.""... no (communication was not effected) ... he responded well to the requests and commands.""yes (pain was significantly less)... he had lot more pain with cleaning (of the wound) than taking the dressing off... He seemed to cope better with VR than without.""... cleaning the dressing in the bathroom made him more anxious, when he saw the wound – felt more pain.""Yes, he was more anxious when VR was not on.""... he felt worse when he was looking at it (the wound) compared to when he was not.""... was more relaxed and concentrated on the game. You could tell that he could feel the pain, but focussed on the game."The perception of the assisting nurses were the children were more cooperative and distracted from the administration of VR, which helped reduce the difficulty in changing the burns dressing compared to when routine analgesia was used by itself. There appeared to be no problem in physically using VR within the environmental constraints of the burns dressing area, and in no instance did VR impede communication with the child.All parents agreed with the positive effects of VR in pain management for their child. They all commented that the child's anxiety level was perceptibly less when using VR, and the child looked forward to playing the VR game. Comments below from the parent interviews support the positive VR effects:"... was a lot calmer and enjoyed the VR.""...absolutely, she did not remember about the last dressing that was taken off. She had to be prompted, to remind her of the game and change of dressing.""... much happier than usual. He reckoned he felt it but I think he did not. He did not show any of the same signs.""...she was not as anxious. Was afraid before hand, but she was all right after the game was switched on. It took her mind, off the pain.""...was smiling while playing the game.""... compared to the medication which left him groggy, disoriented, lost track of time and anxious, I think this (VR) allows the continuity of time and reduces anxiety.""Yes (pain was significantly less) ... probably judging it from yesterday – medication made him worse – uncooperative and pig-headed, compared to when he was playing the game.""Yesterday he was whinging thinking about the dressing change, this morning, when I told him that you were coming; he had a grin on his face..."However, the current game appeared to have a reasonable level of  complexity and engaged the participating children of different age  groups.The above comments were randomly selected from the questionnaire deployed at the completion of each trial .This is the first published randomised clinical trial to our knowledge reporting the use of VR for children with burns. It concurs with the findings of Hoffman et al ,16 who tGiven that the application of VR as a method for pain control in the clinical setting is very simple, the results of this study are encouraging with respect to future use. The present prototype VR game being used appears to be cumbersome due to the number of wires attaching to the laptop and the HMD, but this could be simplified considerably so that the equipment required is simply a console with a trigger and a head mount. Applying this equipment would be as simple as providing medication prior to the dressing change procedure. The equipment is reusable and requires minimal technical knowledge for use. Provided a number of different games were available to cater for different age groups, it could be widely applied, and will allow children to relocate themselves to 'another world' during dressing changes, decreasing their attention to painful stimuli. It was noted by nurses during several trials that communication was never a problem; they were able to instruct the child to change, or assist change in position without any difficulty in a compliant and relatively pain-free manner. On the other hand, children without VR were often distressed and crying in pain, decreasing their ability to listen and cooperate.A number of factors resulted in a small sample size. There were relatively fewer children with burns than anticipated, who fitted the inclusion – exclusion criteria, and who were admitted to the Women's and Children's Hospital, Adelaide, during the data collection period. Several potentially eligible children  had such severe side effects from the medication (particularly drowsiness) that they were not able to participate, and two refused to participate. The reduced sample size limits ability to generalise the results, and a study with a larger sample size may provide better understanding of the usefulness of VR as a treatment adjunct for pain relief.Another potential limitation is that some children were tested more than once – in the first instance immediately after their burn and then subsequently after surgery or during another dressing change later in the healing phase. In these cases, there may have been a learning effect which modified the pain scores, or simply an overall decrease in pain due to healing. It was also noted from the feedback that older children found the game too simple and therefore not as absorbing or distracting as the younger children found it.Finally, waterproofing of the VR equipment would allow data collectors to examine pain responses through an entire dressing change including when subjects had their wound debridement carried out in the bathroom.There appears to be considerable scope for further research into the potential for using VR in the clinical setting. Larger trials could be conducted, using games appropriate for the varying age groups. The next stage would be to test VR alone against pharmacological pain relief, to investigate whether VR is as effective in isolation, and could decrease use of analgesia, thus avoiding the side effects associated with medication. Another avenue of future research would be to investigate the exact mechanisms by which VR assists pain modulation. It is hypothesized that it works by distracting a child's attention from painful stimuli, which in turn reduces the perceived intensity of pain.The author(s) declare that they have no competing interestsDAD was involved with coordination and recruitment of trial  subjects, application of the trial, collection of data and writing up  the paper. KAG was involved with statistical analysis of the data and  supervising the scientific conduction of trials. ALS was a key medical  consultant for the trial subjects and coordinated efforts within the  hospital to recruit trial subjects. SEM was the first contact with the  prospective trial subjects. She identified and provided them with  initial information and got verbal consent from them. She also acted as  a coordinator for all the nursing staff members on the ward  participating in the trial. BAT provided IT support for building and  helped in maintaining the inventory (VR equipment) and the game  software.The pre-publication history for this paper can be accessed here:Pain scale scoringClick here for fileCaregiver interviewClick here for fileNurse interviewClick here for file


Traditionally the rapid origin of megadiverse species flocks of extremely closely related species is explained by the combinatory action of three factors: Disruptive natural selection, disruptive sexual selection and partial isolation by distance. However, recent empirical data and theoretical advances suggest that the diversity of complex species assemblages is based at least partially on the hybridization of numerous ancestral allopatric lineages that formed hybrids upon invasion of new environments. That reticulate speciation within species flocks may occur under sympatric conditions after the primary formation of species has been proposed but not been tested critically.Pungu maclareni.We reconstructed the phylogeny of a complex cichlid species flock confined to the tiny Cameroonian crater lake Barombi Mbo using both mitochondrial and nuclear (AFLP) data. The nuclear phylogeny confirms previous findings which suggested the monophyly and sympatric origin of the flock. However, discordant intra-flock phylogenies reconstructed from mitochondrial and nuclear data suggest strongly that secondary hybridization among lineages that primarily diverged under sympatric conditions had occurred. Using canonical phylogenetic ordination and tree-based tests we infer that hybridization of two ancient lineages resulted in the formation of a new and ecologically highly distinct species, Our findings show that sympatric hybrid speciation is able to contribute significantly to the evolution of complex species assemblages even without the prior formation of hybrids derived from allopatrically differentiated lineages. The scenario may either involve secondary localized hybridization, i.e. hybridization of parapatric  lineages within the geographical range of the primary radiation, or alternatively hybridization of sympatrically diverged lineages. Distinguishing between these two alternatives is central for the understanding of the processes that lead to the evolution of megadiversity. The first alternative predicts that increased species richness due to hybridization is dependent primarily on the spatial scale and the accompanied possibility to establish localized metapopulations. The second alternative predicts that hybridisation can aid the build-up of diversity even under fully sympatric conditions. Several studies, some published, some in preparation support the hybrid swarm origin hypothesis for some species assemblages endemic to comparatively large areas [Recent empirical data and theoretical advances suggest that the diversity of complex species assemblages is based at least partially on the hybridization of numerous ancestral allopatric lineages that formed hybrids upon invasion of new environments -5. A groge areas -5,8 but However, despite increasing evidence for sympatric speciation, uncontested examples remain rare and rarely go beyond the formation of single species-pairs ,16. In aSarotherodon galilaeus. Because the lake's conical basin is only 2.15 km in diameter, because there are no migration-barriers along the shore, and because the lake is isolated from nearby river systems by cataracts of its outflow, allopatric scenarios for the origin and diversification of the flock were ruled out. However, in the light of the aforementioned methodological drawbacks of mitochondrial phylogenies, both the monophyly-hypothesis for the Barombi flock and the relationships among its 11 endemic species are worth to be reevaluated.Until recently the phylogeny of mitochondrial lineages of the cichlid species flock of crater lake Barombi Mbo (Cameroon)  was consThe ability to score thousands of amplified fragment-length polymorphisms (AFLPs) has created a powerful possibility for the phylogenetic reconstruction of rapidly originated species flocks. It has been successfully applied to a limited number of taxa belonging to the Lake Malawi and Lake Victoria haplochromine species flocks and revealed previously undetectable phylogenetic patterns including those supporting the hybrid swarm origin hypothesis ,18,19. IS. galilaeus populations (one to two samples per population). 2191 sites of the alignment were constant, 198 variable characters were parsimony-uninformative and the number of parsimony-informative characters was 164. Empirical base frequencies in this data set were A = 0.2721; C = 0.3271; G = 0.1268; T = 0.2740. Bootstrapped Maximum Parsimony (MP), Maximum Likelihood (ML) and Neighbour Joining (NJ) trees all recovered identical 50%-majority rule consensus-trees  [Pungu haplotypes from Konia took place, while all other clades radiated into several species much later. A 94 sample data set with only cytochrome b and partial proline tRNA sequences  confirmed the previous findings for the Barombi flock and suggests that lineage sorting between the four large clades is complete , but not within Stomatepia ssp., S. caroli and S. linnellii.We obtained a DNA sequence-alignment with 2553 bp including two complete mitochondrial genes, NADH dehydrogenase subunit 2 (ND2) and cytochrome b (cytb), partial proline tRNA as well as from part of the control region from all Barombi species (two samples per species) and relevant Stomatepia and Konia clades , Myaka is sistergroup to the other Sarotherodon subclade consisting of the pelagic species S. caroli and S. linnellii. The Konia clade is resolved as the sistergroup to the rest of the flock. In contrast to the unresolved basal topology in the mitochondrial tree, the two species from Lake Ejagham are resolved as a monophylum which is the sistergroup to the geographically closest S. galilaeus population from the river Cross. An additional data set with 3 selective amplifications but 80 samples recovered only three supraspecific nodes with moderately high bootstrap support, the monophyly of the Barombi flock, Konia and the node containing the rest of the Barombi flock without Pungu. The latter was placed intermediate between the two well supported nodes.The phylogenetic reconstruction based on the same individuals and using 22 restrictive primer combinations with 3489 AFLP size fragments (3004 variable) confirmed the monophyletic origin of the Barombi Mbo flock and the monophyly of the s figure . HoweverPungu maclareni, is the result of speciation by hybridization. By identifying the clades which contain taxa with discordant phylogenies we hypothesized that traces of three ancient hybridization events are still detectable in the multilocus AFLP data. To test for the presence of the respective phylogenetic signal for these three hypothetical ancient syngameons in the large AFLP data set, we used the recently developed method of Canonical Phylogenetic Ordination (CPO) [Both the Shimodaira-Hasegawa and Templeton's test confirmed significantly the difference for the alternative tree topologies for each sequence and AFLP data sets, respectively figure . These don (CPO) . In addiS. galilaeus and lake taxa without S. gal multifasciatus and S. gal "Niger" and (2) the node of the AFLP based hypothesis of the monophyly of the Lake Ejagham sister pair. Of the three hypothetical syngameons, the one uniting Pungu with its potential ancestor clades represented now by the Konia ssp and S. steinbachi and S. lohbergeri contributed significantly to the variation in the AFLP data set, whereas the other two did not.All phylogenetic groups detected either by the mitochondrial or the AFLP data set, or formulated according to the three hypothetical syngameons were coded as phylogenetic variables and their explanatory value for the variance in the AFLP data set was tested table . As a rePungu, a tree-based method as outlined in Seehausen [Pungu resulted in far outlying higher support values for the respective nodes, whereas all other removals resulted in much lower values  based on the well supported phylogeny of mitochondrial haplotypes in the lake, the primary radiation in Barombi Mbo resulted into the almost instantaneous split into four distinct lineages approximately one million years ago. The accumulation of numerous apomorphic characters that support these mitochondrial lineages suggests strongly that they represented reproductively isolated species at that time. Only in an advanced stage of species flock formation and after considerable time had elapsed, their cohesion was broken partially by hybridization events between these lineages. However, according to the chronogram the ancestral mitochondrial clades that contributed, for example, to the hybrid origin of Pungu maclareni, represents an ecologically highly specialized ecotype. Both its peculiar dentition and the accompanying hypertrophic jaw-muscles are unique not only in Barombi Mbo but in cichlids in general [Konia dikume, ranks among the most unusual cichlids as it is the single species which is able to exploit chironimid larvae in the almost oxygen-free deep water due to its extremely high haemoglobin concentration in its blood [Traditionally the rapid origin of megadiverse species flocks of extremely closely related species was explained by the combinatory action of three factors: Disruptive natural selection, disruptive sexual selection and partial isolation by distance -24. Alth general . Accordits blood . In the Sarotherodon galilaeus populations and from all species endemic to crater lakes Barombi Mbo and Ejagham, which are related to S. galilaeus. Oreochromis niloticus and Sarotherodon melanotheron were used as outgroups based on published information and pilot-study data confirming their outgroup status with respect to S. galilaeus and the investigated species flocks.We obtained genetic data from relevant Sarotherodon caroli and S. linnellii. Black adult breeding males of the S. caroli/S. linnellii-phenotype from the deepwater were identified as S. caroli, whereas golden males from the shallow inshore area were identified as S. linnellii. Fin-clips were taken from the right pectoral fin in the field directly after collection and preserved in 96% Ethanol p.A.. Samples from additional specimens belonging to different populations of Sarotherodon galilaeus and outgroups were either collected by UKS in the field in Cameroon or donated by others. Vouchers are deposited in the ichthyological collection of Bavarian State Collection of Zoology (ZSM). Of few S. galilaeus-samples, only photographs of the specimens are available and are deposited in the ZSM, too. Informations about specimens and their species identifications, geographic origin, accession numbers and information about which specimens were sequenced and AFLP typed in different data sets are provided in Adult specimens from Lake Barombi Mbo were collected by UKS during field visits to the lake in 2001 and 2002 and from Lake Ejagham in 1994. Identification to species is according to Trewavas et al.  and was 2 fin tissue with the DNeasy™ Tissue Kit (Qiagen). DNA-quality was visually inspected under UV-light on a 0.8% agarose-gel stained with ethidium bromide. For subsequent AFLP-analysis only samples with a clearly visible high-molecular band were used. DNA-concentration was determined using the VersaFluor-Fluorometer-System (BioRad) using the stain Picogreen® dsDNA Quantitation Kit (Molecular Probes). All samples were adjusted to 60 ng/μl.DNA samples were isolated from approx 10 mm7 for the 5'-end of the control region. PCR was performed by using a PTC 220 DYAD thermocycler (MJ Research) in a 25 μl reaction volume using the Expand PCR system (Roche Diagnostics) with 25 pmol of each primer, 20 pmol of dNTPs, 12.5 pmol MgCl2 and 0.88 units of Taq polymerase. PCR parameters were 94°C for 4 min, 35 cycles with 94°C for 1.5 min, 55°C for 1 min, 72°C for 1.5 min, followed by a final elongation at 72°C for 3 min. PCR products were cleaned by using MinElute PCR purification kit (QIAGEN) and their DNA-concentration adjusted to 100 ng/μl. PCR-Products were then used as templates for cycle-sequencing reaction using the "Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit" (Applied Biosystems) with each of the PCR primers or internal primers. Cycle parameters were the following: 94°C, 2 min; 25 cycles of 94°C, 20 s; 52°C, 10 s; 60°C, 4 min. The sequenced product was filtered through Sephadex-G50 fine (Fluka) packed spin columns (Amersham) to remove unincorporated dye terminators, primers, and salts, and finally dried in a speed-vac. These products were resuspended, electrophoresed and analysed with an ABI PRISM™ 377XL-96 automated sequencer using a 4.25 % polyacrylamid gel (BioRad). Electrophoretic information was transcribed to sequence data using the program Genescan (PE Applied Biosystems).Two different data sets were assembled, one long data set with approx. 2550 bp including the complete NADH dehydrogenase subunit 2 (ND2), the complete cytochrome b (cytb) gene and part of the proline tRNA, and finally, one part of the control region. For this long data set only two samples per species were used, but a short data set with more individuals but only the cytochrome b and the partial proline tRNA genes was generated, too. ND2 was PCR-amplified using the primers "ND2Met" 5'-CATACCCCAAACATGTTGGT-3'"ND2Trp" 5'-GTSGSTTTTCACTCCCGCTTA-3'; a second fragment containing the cytb, proline and threonine tRNAs and the 5'-end of the control region was amplified with the primers "L14725" 5'-TGACTTGAAAAACCATCGTTG and "H16498" 5'-CCTGAAGTAGGAACCAGATG  ; internIndividual sequence files were edited and contigs assembled using Sequence Navigator™ (PE Applied Biosystems). Homologous protein-coding regions  were aligned manually and confirmed by translating DNA data into amino acid sequences in BioEdit . The shoDYE): TC-CAFAM, AT-CAFAM, AC-CCNED, TA-CAFAM, AA-CTFAM; AA-GGJOE; AC-CAFAM; AA-CAFAM; AG-CAFAM; AA-CGJOE; AC-CTFAM; AG-CTFAM; AT-CTFAM; AG-GGJOE; TC-CTFAM; TA-GGJOE; AG-ACNED; AT-ACNED; AG-CCNED; AC-ACNED; AT-CCNED; TA-CGNED. For the short data only the five primer pairs TC-CAFAM, TA-GGJOE, AC-CAFAM, AC-CCNED, AT-CAFAM were used but typed for 80 individuals  with an ABI PRISM™ 377XL-96 sequencer. Fluorescent signals were detected using the GENESCAN software (Applied Biosystems) with internal size standard . The fluorescent threshold was set to 50 units and the correct identification of ROX-marker bands by GENESCAN was checked for all electropherograms.. The program generates aligned spreadsheets from GENESCAN-sized AFLP-data by assigning each sized fragment to a size-class of user-defined distance to the next size-class. Using a spreadsheet routine, fragments were inferred in a second step to be homologous if they differed by no more than 1.00 bp, and if the scoring procedure identified the same size-classes whether scored from small to large size-fragments (forward) or vice versa (backward). Size-classes with inconsistent allocation of fragments according to forward and backward scoring were excluded, as well as adjacent size-classes differing by less than 0.35 bp, which corresponds to the double standard deviation of 0.15 bp of the sequencer [Bands between 100.5 bp and 499.5 bp were scored in a first step for presence or absence using the software Binthere . Non-parametric bootstrapping with 1000  or 100 (ML) pseudoreplicates was used for testing the robustness of the inferred trees. Tree topologies using the HLRT settings under ME were not different from topologies gained with AIC settings (data not shown).The AIC settings were subsequently used for Maximum Likelihood (ML) analyses and to estimate ML distances for minimum evolution (ME) analyses in the program PAUP* 4.0b1.0 (PPC/Altivec)  and for 2 = 701.0154, df = 32, p = 0.001). To date cladogenetic events in the absence of rate constancy, the nonparametric rate smoothing (NPRS) method [A LRT  as imple) method  as imple) method  was usedSarotherodon galilaeus sensu lato (excluding Oreochromis niloticus and Sarotherodon melanotheron). "Pruned" data matrices using only those parsimony informative sites were constructed for Principal Canonical Ordination and homoplasy excess tests (see below) in order to account for noise in the data potentially introduced by the distant outgroup. Pairwise genetic distances were calculated from the binary data-matrix with two different algorithms: One developed by Link et al. [We used PAUP*  to calcuk et al.  as implek et al. , which ik et al. . Trees wk et al.  with unck et al. . Non-parAlternative phylogenetic hypotheses produced as described by tree topologies based on mtDNA and AFLP data were compared with each other and statistically evaluated using either the Shimodaira-Hasegawa LRT  for mitoIn order to test for the presence of a phylogenetic signal that possibly reflects reticulate events in the AFLP-data, two methods were applied.Sarotherodon galilaeus-clade  provided the data-matrix to be tested. Phylogenetic hypotheses derived from mtDNA- and AFLP-analyses as well as hypothetical syngameons as derived from the conflict between the two data-sets were translated into a phylogenetic matrix by assigning binary indicator variables, each coding for the membership of investigated samples to phylogenetic groups  [First, a canonical correspondence analysis (CCA) was performed using CANOCO 4.0 . This megameons) ,54. 9999Sarotherodon steinbachi and S. lohbergeri in the 2355 loci data with n = 16 experiments (each taxon removed once). Analogous support values for the node uniting the Konia eisentrauti and K. dikume were computed with the 530 loci data set with n = 14 experiments, because bootstrap support in the larger data set was always larger than 98.85% and identical runs yielded values differing by more than 1.15 % (data not shown). By reducing the number of loci but increasing the number of samples we obtained a meaningful distribution of bootstrap support values for that node.Second, a tree-based method as outlined in Seehausen  was perfUKS designed the study, carried out the field work, part of the lab work, the major part of the data analysis and wrote the manuscript. BK carried out the major part of the lab work and carried out parts of the data analysis. Both authors read and approved the final manuscript.Sarotherodon melanotheron (not shown)Neighbour Joining phylogram of 94 specimens Neighbour. Joining phylogram calculated from complete sequences of the mitochondrial cytochrome b gene (1141 bp) and the partial proline tRNA gene (71 bp) sequences for 94 specimens based on the GTR+ Γ model and rooted with Click here for filea. The removal of Konia dikume resulted in an increased bootstrap support for the clade uniting Konia and Pungu in the analysis of the 530 loci data; set. b. The removal of S. caroli resulted in increased outside values for the bootstrap support of the b. the Myaka/S. linnellii node and the c. the clade that unites all Barombi taxa with the exclusion of Konia; d. the exclusion of both Myaka and K. eisentrauti increased the bootstrap support for the S. linnellii/S. caroli node; e. Finally, the removal of S. mariae increased the support for the S. pindu/S. mongo split strongly. f. Among the riverine populations of S. galilaeus, the removal of S. gal. "Meme" increased strongly the value for the clade uniting the S. gal. "Cross" specimen with the two endemic species from Lake Ejagham ("Cross clade"); in addition, the exclusion of S. lohbergeri increased the bootstrap value for the same node, albeit weakly.Additional box-plots of bootstrap supports in taxon removal experiments. Box-plots of the distribution of %-bootstrap support values for nodes that yielded additional higher outside or far outside values in taxon removal experiments , Genbank-Accession numbers and representation of specimens in two AFLP-data sets.Click here for file


Immunity against the T cell receptor (TCR) is considered to play a central role in the regulation of experimental allergic encephalomyelitis (EAE), a model system of autoimmune disease characterized by a restricted usage of TCR genes. Methods of specific vaccination against the TCR of pathogenetic T cells have included attenuated T cells and synthetic peptides from the sequence of the TCR. These approaches have led to the concept that anti-idiotypic immunity against antigenic sites of the TCR, which are a key regulatory element in this disease.93ASSDSSNTE101 and 39DMGHGLRLIHYSYDVNSTEKG59).The present study in the Lewis rat used a conventional idiotypic immunization based on antigenized antibodies expressing selected peptide sequences of the Vβ8.2 TCR (68GSLPQKSQRSQDENPVVHF88 in complete Freunds' adjuvant (CFA). Antigenized antibodies induced an anti-idiotypic response against the Vβ8.2 TCR, which was detected by ELISA and flowcytometry. No evidence was obtained of a T cell response against the corresponding Vβ8.2 TCR peptides.The study demonstrates that vaccination with antigenized antibodies markedly attenuates, and in some instances, prevents clinical EAE induced with the encephalitogenic peptide The results indicate that antigenized antibodies expressing conformationally-constrained TCR peptides are a simple means to induce humoral anti-idiotypic immunity against the TCR and to vaccinate against EAE. The study also suggests the possibility to target idiotypic determinants of TCR borne on pathogenetic T cells to vaccinate against disease. Experimental allergic encephalomyelitis (EAE) is an experimentally induced autoimmune disease mediated by T cells. It can be induced in susceptible animals either by immunization with myelin basic protein (MBP) or proteolipid protein PLP, or by immunization with synthetic peptides from the MBP sequence . EAE can+ T lymphocytes bearing an antigen receptor (TCR) variable (V) regions belonging to a limited set of TCR V region gene families -thymidine was added to each well. Cells were harvested onto glass fiber filters and counted on an automatic Beckman LS 6000IC β-counter.Poplyteal, inguinal and paraortic lymph nodes were removed from immunized animals at different times, dissociated and washed in RPMI-1640. Lymph node cells were plated in round-bottom 96-well plates at 2.5 × 1068GSLPQKSQRSQDENPVVHF88 (30 μg/rat) in the anterior paws (25 μl each) in CFA (H37RA 10 mg/ml). Control rats were similarly injected with transfectoma antibody γ1WT or γ2bWT. Rats inoculated with Freunds' adjuvant only served as additional control. Serum samples were collected from the retro-orbital sinus on day 0 before vaccination, day 21 before booster injection, day 28 before EAE induction, and day 50 after recovery from disease. Sera were stored at -20 C until use.Animals were vaccinated with antigenized antibodies (100 μg/rat) in complete Freunds' adjuvant (CFA) divided equally between the posterior paws (25 μl each) and two points in the back subcutaneously. A booster injection (50 μg/rat) in incomplete Freunds' adjuvant (IFA) was given subcutaneously on day 21. EAE was induced on day 28 by immunization with MBP peptide EAE was monitored daily by two operators for clinical signs using the following scale: grade 0 = no appreciable symptoms; grade 1 = tail atony; grade 2 = paraparesis; grade 3 = paraplegia; grade 4 = paraplegia with forelimb weakness, moribund state. Typically symptoms of disease began to appear on day 11–13 from the injection of the encephalitogenic peptide. The Disease Index was calculated according to the formula: [(Maximum Score) × (Duration of Disease) × (Incidence)].Statistical analyses was performed using the Fisher's test.93ASSDSSNTE101 and CDR2 39DMGHGLRLIHYSYDVNSTEKG59 sequences, and were termed γ1TCR-I and γ1TCR-II, respectively  used as a control. Anti-TCR (anti-idiotypic) antibodies were tested using two approaches. In the first case, sera of immunized animals were tested on Vβ8.2 synthetic peptides by ELISA. A weak but distinct response was detected in both instances starting on day 21 or 28 , we engineered an antibody with the same V region (γ2bTCR-I). Rats vaccinated with γ2bTCR-I and subsequently immunized with MBP peptide, were protected only partially compared to rats vaccinated with γ1TCR-I (10.2 vs. 1.9)  Table . All ser-5 M (26). It also binds antigen:antibody complexes formed at molar equivalence with an affinity about 100 fold higher [93ASSDSSNTE101, we would have found similar results. As shown .+ and CD8+ T cells [In this report we demonstrate that the severity of EAE in the Lewis rat can be greatly attenuated, and in some instances completely prevented, by active immunization with antigenized antibodies expressing amino acid sequences of the rat Vβ8.2 gene product. Immunity against synthetic peptides of the TCR has been shown to be effective in preventing or reducing the severity of EAE in the rat -20, sugg T cells -38 as we T cells . A preva T cells  and in t T cells  idiotypi T cells ,42,43 or T cells  have beeHere we show that immunity against idiotopes of antibodies engineered to express TCR peptides is effective in generating anti-idiotypic immunity directed against rat Vβ8.2 TCR gene product. Importantly, this type of immunity protected from EAE. The new approach used herein to induce anti-TCR immunity is based on conventional idiotypic immunization in which antigenized antibodies mimic the immunogenic properties of soluble TCR functioning as a surrogate internal image  in much + T cells suggests that cross-reactive anti-idiotypic antibodies may be very low titer. Alternatively, they may be adsorbed on T cells in vivo precluding their detection in the serum. The first possibility is consistent with the self nature of TCR peptides and a predicted paucity of self reactive clonotypes within the natural B cell repertoire. Interestingly, we noted that the anti-idiotypic response against a non-self peptide expressed in an antigenized antibody [93ASSDSSNTE101 sequence developed flowcytometry-reactive autoantibodies could reflect difference in conformation once the two peptides are embedded in the CDR3 loop of an antigenized antibody. For instance, 93ASSDSSNTE101 could be better surface exposed and more stably expressed 39DMGHGLRLIHYSYDVNSTEKG59. A computer-assisted comparison of hydrophilicity profiles [93ASSDSSNTE101 peptide in the parental. TCR Vβ8.2 gene product and in the antibody V region shows that in both instances the peptide is highly hydrophilic  of cases . We conc93ASSDSSNTE101 but not 39DMGHGLRLIHYSYDVNSTEKG59 reacted immunologically with a synthetic peptide of human CD4 previously described to bind Ig [93ASSDSSNTE101 serum antibodies underscores qualitative differences of the immune response between rats immunized with γ1TCR-I and 1TCR-II, respectively. Thus, it appears as if 93ASSDSSNTE101 induced a different immune response than 39DMGHGLRLIHYSYDVNSTEKG59. Furthermore, since vaccination with γ1TCR-I also promoted greater protection from EAE, it is tempting to speculate that a component of the anti-idiotypic response against γ1TCR-I is associated with protection.Interestingly, rats immunized with the antigenized antibody expressing  bind Ig . BecauseIn conclusion, three points have emerged from this study. First, antigenized antibodies expressing conformationally-constrained loops of the Vβ8.2 TCR can be used as vaccines in the prevention of EAE in the Lewis rat. Our new approach to generate anti-TCR immunity, confirms the relevance of anti-idiotypic regulation in controlling rat EAE ,18,20. SThe authors declare that they have no competing interests


Hypertension is a growing worldwide problem associated with an increased risk of cardiovascular morbidity and mortality. However, the rates of prevalence of hypertension are higher in some populations than others. Although ethnic and genetic factors have been implied in the past to explain this, the environmental influence and psychosocial factors may play a more important role than is widely accepted. Examining the non-genetic influences in future hypertension research may be necessary in order to clearly define the local blood pressure demographics and the global hypertensive disease burden. Hypertension is a common problem, with a consistent and continuous risk of cardiovascular disease and stroke associated with rising blood pressure levels . FurtherThe consequences of poor blood pressure control are huge. As high blood pressure is the most important risk factor for cardiovascular disease, it has been calculated that by achieving the target of 140 mmHg, there would be a reduction of 28–44% in stroke and 20–35% in ischaemic heart disease depending on the age. This would prevent approximately 21400 stroke deaths and 41400 ischaemic heart disease deaths each year – and these translate to approximately 42800 strokes and 82800 ischaemic heart diseases saved, making a total of 125600 events saved per year in the United Kingdom alone [Nonetheless, what is more intriguing and perhaps still not fully explained, is why some populations seem to have a much higher population prevalence of hypertension as compared to others. For instance, the prevalence and incidence of hypertension differs between the non-westernised and westernised populations. Even within the western world, the Afro-Caribbean or African-American black population has a higher prevalence of hypertension and target organ damage related to it, as compared to white Europeans or Americans . DiffereBMC Medicine, address the issue of whether there is a truly genetic predisposition or perhaps an environmental influence is to blame for higher rates of prevalence of hypertension seen in some of these ethnic populations. In a well-designed pooled analysis, incorporating eight studies involving 8 white and 3 black populations from the North American, European and African populations – a dataset of nearly 85,000 patients – Cooper et al [Cooper et al , writinger et al  examinedper se together with the unhealthy lifestyle and diet in the western world increase the risk of hypertension, compared to the rural, 'low stress', healthier lifestyle and dietary habits in Africa? Perhaps the genotype of black subjects was not idealised for the 'pro-hypertension' environment of the western world, leading to the greater risk of developing hypertension amongst blacks in the western world. This 'genetic predisposition' of certain ethnic groups, coupled with the 'wrong' environment, leads to an unhealthy combination that predisposes to cardiovascular disease [Does this take us back to the drawing board? Perhaps environmental factors do play a more major role in developing hypertension than is widely accepted. Indeed, does urbanisation  disease . However disease . Therefo disease . SimilarCan this 'wrong genotype in the wrong environment' hypothesis be applied to hypertension in black African-Americans? Efforts have already been made to understand the reasons behind the higher prevalence of hypertension in African Americans, with the underlying assumption that there may a genetically determined predisposition as compared to their white counterparts; however, no convincing data are available . This maNumerous potential explanations for the higher prevalence of hypertension in blacks have been proposed. Genetic mechanisms have been used to explain familial aggregation of hypertension in Jamaican blacks and the intra-class correlation of systolic blood pressures amongst black twins ,14. Low Notwithstanding the shortcomings of a retrospective, pooled study, with varying criteria for inclusion, and the intra-, inter- and cross-study observational errors, Cooper et al  have shoEvidence that environmental and particularly psychosocial factors are important in the development of hypertension also comes from a series of epidemiological studies conducted in the early part of the last century, which have shown that urbanisation and adoption of a westernised life style leads to blood pressure rises. Many of these studies have been conducted in Africa, where the rural populace has a relatively low prevalence of hypertension. For example, primitive black populations living in more frugal circumstances in rural Africa have been shown to have low blood pressures. Evidence suggests that there are some populations that exist who are naïve to hypertension and related morbidity. Interestingly, their blood pressures hardly rise with increasing age, a common response to age in many other (urbanised?) communities -18. The Migration, not withstanding the complexity of the studies in these populations, has been shown to significantly affect blood pressures. A study examining the migrant islanders into New Zealand showed raised population blood pressures, as well as an increased slope of the age-blood pressure relationship . The KenThe issue of the influence of colour of the skin to blood pressures is even more complex. On the one hand, studies in America have shown relationship between the dark skin and blood pressures, leading to some suggesting that the link is genetic. In contrast, some argue that it is a manifestation of the stress and social pressure of having a dark skin that causes this. In Cuba, for example, where communist principles are considered to have broken the racial barriers, the ethnic differences in blood pressures were shown to be small, supporting the latter argument . In addiThus, the process by which a society becomes more economically advanced or "developed" seems to be associated closely with rates of hypertension prevalence. Indeed, lifestyle and dietary changes related to the so-called "development" seem linked to the prevalence of hypertension. In the INTERHEART population case-control study , which wThere remain many uncertainties to the relative importance and contribution of environmental versus genetic influences on the development of blood pressure – there is more than likely an influence from both. However, there is now evidence to necessitate increased attention in examining the non-genetic influences on blood pressure, a neglected area of hypertensive research but perhaps a goldmine for establishing causal influences. As stated earlier, the future of hypertension research should focus on more standardised and comparable protocols, with comparable designs in data collection. Multi-centric data collection with a view to establishing local or national blood pressure demographics is crucial for the formal assessment of the global hypertension burden and the implementation of cost-effective primary preventive measures.The pre-publication history for this paper can be accessed here: