In spite of continuous improvements in device design and applications, the profound use of heart pump has been limited because of its high price. The available clinically applied heart pump costs mostly about 100 thousands US Dollars. The author has since long tried to develop a heart pump costing only 1000 Dollars for recovery or bridge to heart transplantation therapies. The device is a radially driven centrifugal pump with a brush-less DC motor and a streamlined impeller. Its bearing is rolling bearing using 4 to 6 needles, manufactured by special wear-proof polythene with super-high-molecular weight, thus the service life achieves more than 10 years. To avoid thrombus formation, a special purge system is introduced to the bearing, allowing the saline with heparin to be infused through the bearing into the pump. The bearing, therefore, keeps working in the saline, and absolutely no thrombus will be formed along the bearing. Animal experiments demonstrated that a 30 mL fluid infusion per hour is enough to prevent thrombus formation. With these improvements, the impeller pump has continuously run for 14 months in the laboratory, and no bearing wear can be measured. The device, weighing 150 g, is fully implantable, consumes approximately 9.6 W, delivers a 9Lmin-1 blood flow against a 120 mmHg mean pressure, and reaches a highest total efficiency of 24.7% for the motor (including the controller) and the pump. The device has been used in animal experiments together with an American artificial lung for more than one month in the University of Texas and also in human trials in the Taiwan University. The R&D of artificial heart has over 50 years’ history. In the first 25 years, the diaphragm pumps imitating the function and mechanism of the natural heart had demonstrated the feasibility of man-made mechanical pumps in support or replacement of both left and right ventricles partly or totally. As soon as people realized that the diaphragm pump is too complicated, the simple rotary pump has been investigated extensively in the recent 25 years. Now an axial pump has worked on a patient for more than 7 years . Sooner In spite of the technical progress in heart assist pump, its profound application has been limited because it costs about 100 k US Dollars. Most patients especially from the developing countries need a sufficient and reliable VAD but can not use such expensive device. The author has tried to develop a heart pump costing only 1000 dollars and has succeeded in the recent years . This pa1) is a schematic drawing of the one thousand dollar blood pump. It consists of a motor and an impeller (2) in a pump housing (1). The motor has rotor magnets (3) and motor coil (4) winded on an iron core. The impeller is fixed on the rotor, which is bore by 4 to 6 needles (6) onto a stick screwed on the stator. The structure of the needle bearing is shown in Fig. (2). It’s well known that the rolling friction is much smaller than the sliding friction and that the needle material of polythene with super-high-molecular weight has excellent anti-wear property, the service life achieves thereafter over 10 years.Fig.  is devised, allowing the saline with heparin to be infused through the bearing into the pump. The bearing, therefore, keeps working in the saline, and absolutely no thrombus will be formed. Animal experiments demonstrated that a 30 ml fluid infusion per hour is enough to prevent thrombus formation.3) exhibits this elegant pump which had once continuously run for 14 months in the laboratory, and no bearing wear could be measured. The device, weighing 150 g, is fully implantable, consumes approximately 9.6 watts power, delivers a 9L/min blood flow against a 120 mm Hg mean pressure, and reaches a highest total efficiency of 24.7% for the motor (including the controller) and pump. The device has been used in animal experiments together with an American artificial lung for more than one month [4) and also in human trials in the Taiwan University [Fig.  exhibitsA simple, implantable, durable and reliable left ventricular assist device costing only one thousand US Dollars is presented, which may solve the present problems that hundreds and thousands patients, especially in developing countries, need a heart pump but cannot afford it due to its high costs. The author is ready to cooperate in further development of the device to be a clinically applicable device finally.


Neck and shoulder complaints are common among employees in sedentary occupations characterized by intensive computer use. Specific strength training is a promising type of physical exercise for relieving neck and shoulder pain in office workers. However, the optimal combination of frequency and exercise duration, as well as the importance of exercise supervision, is unknown. The VIMS study investigates in a cluster randomized controlled design the effectiveness of different time wise combinations of specific strength training with identical accumulated volume, and the relevance of training supervision for safe and effective training.A cluster randomized controlled trial of 20 weeks duration where employed office workers are randomized to 1 × 60 min, 3 × 20 min, 9 × 7 min per week of specific strength training with training supervision, to 3 × 20 min per week of specific strength training with a minimal amount of training supervision, or to a reference group without training. A questionnaire will be sent to 2000 employees in jobs characterized by intensive computer work. Employees with cardiovascular disease, trauma, hypertension, or serious chronic disease will be excluded. The main outcome measure is pain in the neck and shoulders at week 20.The trial is registered at ClinicalTrials.gov, number NCT01027390. Musculoskeletal disorders cause poor work ability and lost working days , and conPhysical exercise is a cornerstone in health and well-being . An incrInternational health guidelines recommend adults to perform at least 30 minutes of moderate physical activity 5 days per week for general health . While tMost studies investigating the effect of exercise for neck and shoulder pain have used supervision by experienced training instructors or physiotherapists -16. HoweThis study - Work place Adjusted Intelligent physical exercise reducing Musculoskeletal pain in Shoulder and neck (VIMS) - investigates in a cluster randomized controlled design1) the effect of different time wise combinations of training with identical accumulated duration, in order to elucidate possible ranges of training effectiveness and flexibility2) the relevance of training supervision for safe and effective training, in order to minimize expenses for workplace physical exercise training.The concept of 'intelligent physical exercise' is to balance the physiological capacity relatively to occupational exposure, tailor the exercise to individual capacities and disorders, allow for flexibility and interest for the participant, and to be as cost-effective for the company as possible.We are currently conducting a cluster randomized controlled trial in Denmark. The trial began in February 2010 and ends in June 2010. The participants were recruited from 12 geographically different units located in all major cities throughout Denmark balanced according to the population density with around half in the Copenhagen area and half in other parts of Denmark. The criteria were that the sites should be distributed across the country, they should be so large that it was possible to randomize naturally occurring clusters to five groups and the total number of employees in the sites should be around 2000 persons.All of the participants gave their written consent to participate in the study. The local ethics committee approved the study protocol (H-C-2008-103), which qualified for registration in the ClinicalTrials.gov, number NCT01027390Figure Exclusion criteria were (i) hypertension  or cardiovascular diseases, (ii) symptomatic herniated disc or severe disorders of the cervical spine, (iii) postoperative conditions in the neck and shoulder region, (iv) history of severe trauma, and (v) pregnancy, (vi) or other serious disease. Prospective participants had to work at least half of their working hours in an office environment to be included. The remaining 573 participants were randomly allocated at the cluster level to five groups as described below. Table Work place physical exercise has implicit social elements of both getting together, competition and group pressure. For the intervention to capture these elements, we used cluster as the unit of randomization. The clusters were naturally occurring groups of employees, thereby also minimizing contamination between clusters. To help ensure the comparability of the 4 intervention groups and the reference group, geographical sites were categorized into 13 strata. Each stratum was divided into 5 naturally occurring clusters. Building, floor, department and the size of clusters were taken into account when defining the clusters in order to attain intervention and reference groups of similar sizes in each stratum. Strata and clusters were formed by authors MZ, LA and GS. In two of the strata forming of clusters entailed individual randomization into two clusters. This individual randomization was performed by a data manger when stratum and cluster affiliation was attached to the participants.Within each stratum the clusters were randomized to group (group was sampled with replacement and assigned to the cluster). Randomization was performed by author HF using a SAS macro based on the RANUNI function. In more details: Strata were numbered consecutively  and clusters numbered consecutively with strata . Within each stratum, the clusters were sorted according to a random number and assigned the color codes red, green, blue yellow white (a code for the 4 interventions and the reference group) in succession and cyclically. To minimize imbalance over several strata with a number of clusters no divisible by five, the SAS macro was programmed to start the cyclic assignment by assigning one of the colors at random (with probability 1/5). As the formation of clusters resulted in exactly five clusters in each stratum, this element of the macro was redundant.When the training schedules and premises were sent out, just before the start of the intervention, it appeared from the enquiries that a particular subgroup of participants had misunderstood the slightly ambiguous question concerning geographical site. The wrongly placed 43 individuals were identified by comparing questionnaire responses to staff records. Before the intervention commenced the individuals were re-allocated according to the following principles wherever possible:▪ From the response to questions concerning departmental affiliation and floor of the building the true cluster was determined. The respondent was re-allocated to the treatment corresponding to that cluster.▪ When ambiguity with respect to determining the right re-allocation cluster, a cluster allocation resulting in no change in treatment group was preferred.▪ If the respondent belonged in one of the clusters detemined by individual randomisation, the cluster re-allocation was determined by flip of a coin.The four training-groups performed the same total amount of exercises and repetitions per week - i.e. an equal training volume - for a total of one hour per week for 20 weeks during working-hours. Experienced instructors supervised half of the training sessions three of the groups. The first group trained for 1 hour once a week , the second group trained 20 minutes 3 times a week  and the third group trained 7 minutes 9 times a week . Group four  followed the same program as 3MS, but received supervision only during the initial week. The reference group was not offered any physical training, but replied to the same questionnaires as the intervention-groups.The intervention-groups performed specific strength training with 5 different dumbbell exercises, as shown in figure 1. Front raise: From a neutral starting position the participant lifts one arm at a time to 90 degrees shoulder flexion, and 90 degrees internal rotation. The elbows are slightly flexed (~5°) during the entire range of motion.2. Lateral raise: the participant is standing with arms in neutral starting position and the elbows are in a static slightly flexed position (~5°). The participant lifts both arms to 90 degrees shoulder abduction and 30 degrees horizontal flexion.3. Reverse flies: The participant is sitting bent over forward with the back straight and arms hanging. The arms are raised bilaterally, while keeping the elbows in a static slightly flexed position (~5°), until the upper arms are horizontal.4. Shrugs: The participant is standing erect with arms to the side and elevates the shoulders as high as possible in a maximal shrug.5. Wrist extension: sitting with the forearm pronated on a support. From full palmar flexion the participant moves the wrist to full dorsal flexionParticipants performed exercises in a rotating manner to optimally increase training load, and rested 1-2 minutes between sets . Each trDuring the 20-week intervention training loads were progressively increased according to the principle of periodization and progressive overload. Table If a participant experienced incidence of joint pain or the like during a specific exercise, we asked them to adjust the exercise as follows: First, the instructor asked the participant to externally rotate the arms, slightly alter the path of the arms or reduce the range of movement during the exercise. Then, the participant reduced the training load of the exercise (e.g. a reduction of 1 kg). If this did not help, the participant reduced the number of sets, and finally reduced training frequency. If none of these adjustments had the desired effect, the exercise was removed from the participant's training program for at least 1 week.Compliance to training is challenging. Workplace interventions have typically reported low to moderate compliance ,24. In tWe applied a structured email-based questionnaire including e.g. the Standardized Nordic questionnaire for musculoskeletal disorders , the IntMusculoskeletal pain symptoms of the neck, shoulder, arm, hand, and back were evaluated using validated scales concerning both intensity and duration of symptoms. Participants replied to the questions "How many days have you had trouble in [body part] during the last three months?"  for symptom duration, and "On average, how intense was your pain in [body part] during the last three months on a 0-9 scale?" (where 0 means no complaints and 9 means pain as bad as it can be) for symptom intensity. Answers to the question that concerned symptom duration were recoded as follows: 0 days = 0, 1-7 days = 4, 8-30 days = 19, >30 days = 60, everyday = 90. Illustrations from the Nordic questionnaire defined the respective body regions [ regions . FurtherThe level of physical activity were assessed using the International Physical Activity Questionnaire (IPAQ) [e (IPAQ) . Total pSelf-efficacy in relation to barriers towards physical activity was assessed by a questionnaire [ionnaire ,28. E.g.Stages of change in relation to physical activity was in this study assessed by a questionnaire originally presented by Marcus and colleagues in 1992 [ in 1992  but furt in 1992 -32. QuesProductivity was rated on an 11-step ordinal scale: "How do you perceive your overall productivity the last 4 weeks?" The rating went from 0 (the worst a worker could do) to 10 (the best a worker in the same job could do) [ould do) Testing of physical capacity was performed at the beginning, midways, and at the end of the 20-week intervention in all five groups.Maximal muscle strength was assessed by a repetition maximum (RM) test at baseline, at midterm (10 weeks) and at the end of the intervention (20 weeks). We used the bilateral lateral raise exercise . The participant stood with the back against a wall, and performed 1 repetition with 1 kg dumbbells and held the top-position for 1-2 seconds, waited 30 seconds, and then one repetition with 2 kg dumbbells. Using this procedure, the examiner added 1 kg until the participant was unable to lift and hold the dumbbell properly. The maximal weight lifted with acceptable technique was noted as 1 RM.Strength-endurance was assessed with a submaximal load. Using the same exercise as described above, the participant performed as many repetitions as possible with a load of 1 kg less than the 1 RM load. During each repetition the participant held the top-position for 1 second. The participant used the same load during midterm and end of intervention as the one used during the baseline test.The primary outcome is change in pain of the neck and shoulders at 20 weeks. Secondary outcomes include the other measures mentioned above. We plan to analyze the main data according to the intention-to-treat principle. Secondly, we will also analyze data per protocol. We will use repeated measures analysis of variance. Our design allows us to compare both the effect of different combination of duration and frequency and the effect of training supervision figure . The fol1) There is no difference between the groups 1WS, 3WS, 9WS and reference for the change in neck/shoulder pain from baseline to week 20.2) There is no difference between the groups 3WS, 3MS and reference for the change in neck/shoulder pain from baseline to week 20.Power analyses performed prior to the study showed that - to reject the null-hypothesis of equality - we should include 150 participants per group  for 80% power to detect a 10% change in pain between groups.The authors declare that they have no competing interests.GS, MZ, OM, LA and MTP were responsible for the research design. LA drafted the paper, and all co-authors made significant contributions to drafting the protocol. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/11/173/prepub


Individuals with osteoporosis and recent vertebral fractures suffer from pain and impaired health-related quality of life (HRQL). To determine whether patients with osteoporosis treated with teriparatide experienced improvement in HRQL and pain symptoms after several months of therapy.We retrospectively studied a sample of osteoporosis patients treated with teriparatide in a Canadian rheumatology practice. We included patients that received teriparatide therapy with baseline and follow-up Mini-Osteoporosis Quality of Life Questionnaire (OQLQ) data. Follow-up data was measured at three or six months. We used a paired Student's t-test to compare baseline and follow-up measurements for each of the questionnaire's ten questions (five domains). Statistical analysis was also repeated to only include patients who suffered a prior vertebral fracture.57 patients were included in the study, including 47 women. The mean age was 63.8 years (standard deviation 12.1 years). About sixty five percent (37/57) had previously sustained one or more osteoporotic fractures and about 38.6% (22/57) had suffered a prior vertebral fracture. About 44% (25/57) of individuals were taking one or more types of pain medications regularly prior to starting therapy. At follow-up, significant improvements were observed in the OQLQ domains of pain symptoms. This was seen when all patients on teriparatide were included, and also when only patients with prior vertebral fractures were included. There was also an improvement in emotional functioning, relating to fear of falling at 3 months follow-up (p = 0.019). Respondents also reported improvement in the domain of activities of daily living, relating to vacuuming at 6 months follow-up (p = 0.036), and an improvement in the leisure domain, relating to ease of traveling in the prior vertebral fracture population at 3 months follow-up (p = 0.012). However, there was no significant improvement observed in the domains of physical functioning. Participants also reported a decrease in need for pain medications, with 26% (15/57) requiring analgesics at the time of follow-up.Teriparatide use may be associated with improvements in HRQL in osteoporosis patients, in particular alleviation of pain symptoms. These results were especially evident in patients with a history of vertebral fractures. These findings should be confirmed in larger prospective studies with a suitable control group. Osteoporosis is a disease leading to progressive decreases in bone mineral density, decreased bone strength and increased risk of skeletal fractures . ApproxiConventional treatments for osteoporosis, including bisphosphonates, selective estrogen receptor modulators (SERMs), calcitonin and estrogen, have been shown to reduce the rate of bone resorption and preserve bone mass . HoweverIn the FPT trial comparing the effect of Teriparatide 20 μg/day to placebo in post-menopausal women, the incidence of back pain was 17% in the treatment group, and 23% in the placebo group . TeriparThe goal of this study is to determine whether patients in every day clinical practice with osteoporosis, treated with teriparatide, experienced improvement in HRQL and pain symptoms after several months of therapy in a clinic setting. Measuring only pain scores for these patients would be insufficient, because aside from acute and chronic back pain, patients with vertebral fractures also suffer from impaired activities of daily living, anxiety and constant fear about falling and suffering another fracture . A folloWe conducted a review of osteoporosis patients who had been placed on teriparatide therapy in a Canadian rheumatology practice. Two reviewers abstracted data on demographic information, pain medications, previous fracture history, bone mineral density and health related quality of life (HRQL). In order to be eligible for the study, participants must have completed a baseline and follow-up mini-OQLQ questionnaire prior and subsequent to commencement of teriparatide therapy (at 3 or 6 months) Figure .HRQL was assessed using the mini-OQLQ , which wAs mentioned earlier, this is an exploratory study whose sample size is primarily determined by the available data. The results of this study will provide us with some further insight about the potential effect of teriparatide on health-related quality of life in primary care of patients with osteoporosis which can be explored further in major study. All patients with a follow-up visit at either 3 months or 6 months were compliant with their treatment.Descriptive data were reported as means (standard deviations [SD]) or median [minimum [min] – maximum [max]) for continuous or discrete variables and count (percentage) for categorical variables. We used the paired Student's T-tests to compare scores on questions of the mini OQLQ prior to and during teriparatide treatment. Analysis was performed comparing the baseline scores to both the follow-up results after 3 and 6 months of therapy. Patients with a prior vertebral fracture were separated and reanalyzed to assess if teriparatide had a greater effect on those with a pre-existing vertebral fracture and/or back pain. We used normal probability plots to assess the Normality assumption. The criterion for statistical significance was set apriori at alpha = 0.05. We used SPSS 12.0 for all statistical analyses.The study sample included 57 participants, with 82.5% (n = 47) being female Table . The meaThe mini-ORQL can be divided into five distinct domains relating to HRQ: symptoms , emotional function Q3, Q4), physical function , activities of daily living , and leisure . Each question was analyzed individually and grouped according to the domains that they represent . For patients with 3 month follow-up data, the baseline data for Q1  was reduced in follow-up data  (p = 0.028), and for patients with 6 month data, the baseline values of  was reduced to  (p = 0.039). Similarly baseline data for Q2 ; was lower in the 6 month follow-up data ) (p = 0.022). These results were also evident when only patients with only prior vertebral fractures were included. At 3 month follow up, Q1 decreased from  to  (p = 0.005) and at 6 month follow-up, Q1 decreased from  to  (p = 0.007). Similarly after 6 months, Q2 also decreased from  to  (p = 0.009) for the vertebral fracture group.The questions addressing emotional functioning also demonstrated a significant improvement in Q4. The baseline for Q4  was lower after 6 months of therapy  (p = 0.019). However, this was not evident when only considering patients with prior vertebral fractures. Q3 did not reveal any significant improvement after initiating teriparatide.Data for the physical functioning domain did not show a significant improvement in either questions, and there was no difference when only considering patients with prior vertebral fractures.Question 7 which assessed the difficulty of vacuuming showed a significant improvement at the 6 month follow-up point  to  (p = 0.036) but there was no significant. However, this change was not evident when only considering patients with prior vertebral fractures. Question 8 did not reveal and significant improvement.Question 9, which assessed the patient's difficulty in travelling showed a significant improvement after 3 months of therapy in the vertebral fracture patients. The scores decreased from  to  (p = 0.012). Q10 did not reveal any significant improvement after initiating teriparatide.Vertebral fractures are an important and common cause of morbidity in osteoporotic patients . VertebrAnalysis of our HRQL data in our patients' pre and post-teriparatide therapy revealed statistically significant improvements predominantly in the domain of symptoms, but there were also improvements in the domains of emotional functioning, ADLS, and leisure. The symptoms domain inquired about discomfort/distress related to pain, while the emotional functioning domain addressed the patients' fears of two major complications, fractures and falling. A reduction in the symptoms domain indicates that patients who received teriparatide therapy went on to experience less pain than prior to therapy. In addition, there was a decrease in patients requiring analgesics after treatment  How much distress or discomfort have you had because of pain in the last two weeks?2) How much distress or discomfort you had in last two weeks because it had been painful to stand for a long time3) How often in the last two weeks have you felt afraid of fractures?4) How often in the last two weeks have you felt afraid of falling?5) How difficult has it been for you to lift things in the last two weeks?6) How difficult has it been for you to carry things in the last two weeks, because of back problems due to osteoporosis?7) How difficult has it been for you to vaccum in the last two weeks?8) How difficult has it been for you to housework in the last two weeks?9) How difficult has it been for you to travel in the last two weeks?10) How difficult has it been for you to take the type of vacation or holiday you enjoy because of your back problems due to osteoporosisThe pre-publication history for this paper can be accessed here:


Caenorhabditis elegans virtually all progeny produced by hermaphrodite self-fertilization is hermaphrodite while 50% of the progeny that results from cross-fertilization by a male is male. In the standard laboratory wild type strain N2 males disappear rapidly from populations. This is not the case in some other wild type isolates of C. elegans, among them the Hawaiian strain CB4856.In the androdioecious nematode We determined the kinetics of the loss of males over time for multiple population sizes and wild isolates and found significant differences. We performed systematic inter- and intra-strain crosses with N2 and CB4856 and show that the males and the hermaphrodites contribute to the difference in male maintenance between these two strains. In particular, CB4856 males obtained a higher number of successful copulations than N2 males and sired correspondingly more cross-progeny. On the other hand, N2 hermaphrodites produced a higher number of self-progeny, both when singly mated and when not mated.These two differences have the potential to explain the observed variation in male persistence, since they should lead to a predominance of self-progeny (and thus hermaphrodites) in N2 and, at the same time, a high proportion of cross-progeny  in CB4856. Caenorhabditis elegans is a facultative hermaphrodite that reproduces either by virtue of self-fertilization or cross-breeding with a male (androdioecious reproductive system). The hermaphrodites are somatically female but produce a limited number of sperm during their late larval development before switching to the production of eggs. The sperm is stored in the spermatheca and can be used to fertilize the newly formed eggs. Except for a very few males (around 0.2% in the standard laboratory strain N2) that arise spontaneously as the result of X chromosome non-disjunction, the entire self-progeny is hermaphroditic. Males can mate with hermaphrodites and give rise to 50% males in the cross-progeny. Cross-fertilization is not possible among hermaphrodites . Tak. TakCaen elegans . Interes elegans . Similar elegans -22.C. elegans isolates from different parts of the world using a variety of molecular markers such as microsatellites, AFLPs, or DNA sequence polymorphisms. They unanimously demonstrate that outcrossing does occur, but that it is usually extremely rare [-5 up to 0.02 [An alternative albeit indirect route to assess the function of males is to infer outcrossing rates in natural populations. Several recent studies analysed new ely rare -27. The ely rare , whereas to 0.02 -25. Cons to 0.02 ,29,30.In the first part of this publication we describe the decline of the proportion of males in eight different natural isolates under standard laboratory conditions. The fact that males are lost at very different rates even if the strains are maintained under the same conditions indicates that the difference is genetically determined and is therefore a putatively selectable trait. In the second part we evaluate the possible reasons for the difference in male persistence between the two common laboratory strains N2 and CB4856. Several behavioral and physiological factors could account for this difference, for example: i) the mating efficiency of the males, ii) the mating efficiency of the hermaphrodites , iii) the competitive advantage of the male derived sperm, iv) the number of sperm transferred, v) the difference between the maximum number of progeny a hermaphrodite can produce with and without mating. In order to address these points, we performed a systematic analysis of intra- and inter-strain crosses between N2 and CB4856- to our knowledge for the very first time in this context. Our results indicate that CB4856 males are capable of mating successfully with more hermaphrodites than N2 males and that N2 hermaphrodites produced a higher number of  self-progeny even after mating. Both these effects result in a higher proportion of males in the next generation for CB4856 if compared with N2, thus potentially explaining male persistence in the former but not the latter strain.C. elegans was cultured as described in [Escherichia. coli strain OP50 food bacteria and M9 buffer is also described in [ribed in . The preribed in .E. coli (OP50) culture such that the plates contained a small round dot of bacteria in the center.Mating plates: 6 cm NGM plates were seeded with 30 μl of an All experiments were done in an air-conditioned room at a temperature of 21 ± 1°C and 40% humidity. To minimize fluctuations of physical conditions plates were kept in boxes, randomized in piles that were placed evenly distributed within the boxes.N2: Standard laboratory wild type strain, isolated in Bristol, UKCB4856: Standard polymorphic mapping strain, isolated in Hawaii.AB1: isolate from AustraliaJU258: isolate from MadeiraMY1, MY15, MY18, RC301: isolates from GermanyCaenorhabditis Genetics Center at the University of Minnesota [All strains are available from the innesota .C. elegans isolates and in different population sizes. For each strain, we set up several crosses using a male:hermaphrodite ratio of 2:1. These crosses yielded populations with a gender ratio of approximately 1:1. For each strain, the populations were mixed four days after setting up the crosses and a defined number of individuals (population size) was randomly chosen and transferred onto NGM plates for the experiment (day 0 of the experiment). These experimental populations were all treated as follows: After three days adult males and hermaphrodites were counted . On the next day the population was reduced to the original population size and transferred to a new plate . Three days later, adult males and hermaphrodites were counted again, followed by population size reduction and worm transfer one day later, as above. This whole procedure was repeated for a total of eight times . All male maintenance assays were done on 9 cm NGM plates, seeded with 1 ml of E. coli OP50 culture. Two sets of experiments were performed: i) two replica runs per strain and population size were done in parallel for strains N2, CB4856, AB1, JU258, MY1, MY15, MY18 and RC301 using population sizes of 75 and 150; and ii) five replica runs per strain and population size were done in parallel for N2 and CB4856 using population sizes of 40, 70, 100 and 150. In this context, one important objective was to evaluate the effect of different population sizes on male persistance. The exact numbers used (e.g. 75 versus 150) were chosen arbitrarily.This assay served to determine the persistence of males over time in different natural The plates were placed under a dissecting microscope and searched systematically always using the same search path with the help of a grid, which was positioned below the plates. The first 100 to 120 adults encountered were then used to determine the number of adult hermaphrodites and adult males. Note that this is equivalent to a random choice of individuals. Only hermaphrodites with developing embryos in the uterus were counted as adult hermaphrodites. This may have lead to a slight underestimation of the number of hermaphrodites.Worms were washed off plates with M9 buffer and counted without paying attention to the developmental stage or sex of the worms. The volume that was expected to contain the desired number of worms was transferred onto a new plate. Thus, the populations always consisted of mixed generations, so that the effective reproductive population was smaller than the actual number of animals.This assay served to evaluate the mating efficiency of males in terms of mated hermaphrodites, total offspring produced per male and also cross- as well as self-progeny produced per mated hermaphrodite. A single male was confronted with an excess of hermaphrodites, so that it could mate as often as possible. In a pilot experiment, 14 hermaphrodites were found to be sufficient to ensure that the male would never come close to mating with all of them. In fact, during the main experiment the highest number of mated hermaphrodites per plate was 9.E. coli OP50. The hermaphrodites were transferred to new plates every 24 h. After three days the progeny was counted or, if the number of plates was too high to be processed immediately, equal numbers of plates from both treatments were put at 4°C and counted within a few days. This step allowed us to do more replicas in parallel and have all plates scored by the same person, in order to avoid possible observer biases. We did not observe any lethality in response to the cooling step. A hermaphrodite was considered to be mated when more than one male was found among the progeny (successful mating event). The number of cross progeny per mated hermaphrodite was estimated as twice the number of male progeny. Since it was not feasible to do all crosses in parallel, we used an incomplete block design, where two different crosses were set up in parallel and different pairs of parallel crosses were assayed in four experimental runs: i) N2 males crossed with N2 and with CB4856 hermaphrodites; ii) CB4856 males crossed with N2 and with CB4856 hermaphrodites; iii) N2 and CB4856 males crossed with N2 hermaphrodites; iv) N2 and CB4856 males crossed with CB4856 hermaphrodites.Mating plates were prepared four days prior to the experiment. One male and 14 young adult hermaphrodites were placed on mating plates. Every 24 hours the male was moved to a new mating plate with 14 young adult hermaphrodites until no more successful mating was observed. Hermaphrodites, which were exposed to males, were placed individually on NGM plates seeded with 200 μl These two assays were used to characterize in more detail the time required by males until first contact with a hermaphrodite and first spicule insertion (One-hour assay) and also the number of contacts with hermaphrodites as well as spicule insertions over a nine hour period (Nine-hours assay). In both assays, we used the same general conditions as in the male mating efficiency assay (particularly as to usage of 14 hermaphrodites), in order to permit comparison of results.L4 males and L4 hermaphrodites were transferred to separate plates one day prior to testing in order to obtain virgin adult animals. At the beginning of the experiment, one male was placed together with 14 hermaphrodites on a mating plate. We then measured the time until the male touched a hermaphrodite and showed mating behavior (first contact) and until the first time the spicule was inserted (spicule insertion). Observations were terminated after spicule insertion or, if these did not occur, after one hour.14 L4 hermaphrodites per plate were placed on mating plates one day prior to the experiment. At the same time L4 males were collected and placed on a plate without hermaphrodites. To start the experiment single males were transferred onto the mating plates with the 14 hermaphrodites. Within 9 hours, the plates were inspected 14 times . At every inspection, the male was scored as either having no contact with a hermaphrodite, being in contact with a hermaphrodite, or having its spicule inserted.This assay served to determine the number of offspring produced trough self-fertilization in the absence of males for two natural isolates, N2 and CB4856. Young adult hermaphrodites, which had no developing embryos in the uterus yet, were placed individually onto plates and moved to new plates at least once every day until they stopped laying eggs. Three days after the removal of the mother the progeny on every plate was counted and summed up to total numbers.This assay was used to test how more frequent mating affects the hermaphrodite's production of self- as well as cross-progeny. Mating plates were prepared four days prior to the assay. One L4 hermaphrodite was placed together with 1, 3, 6 or 12 males onto a mating plate. The hermaphrodite was transferred onto a new mating plate with new young males every day until it stopped laying eggs. The old males were removed from the plates. After one day, additional food bacteria (OP50) were added to the plates and hermaphrodites and males were counted two days later, when they were young adults.C. elegans strain as fixed predictors. The male proportion at specific time points was additionally examined with a Wilcoxon sign rank test. Variation in the number of mated hermaphrodites, the number of offspring per male, and the self- and cross-progeny per mated hermaphrodite was assessed with a General linear model, using an incomplete block design, including male strain, hermaphrodite strain and the interaction between them as fixed factors and experimental block as a random factor. In case of significant interaction terms, significant differences among groups were evaluated with Tukey HSD posthoc tests. Variation in offspring number per repeatedly mated hermaphrodite was tested with an ANOVA, using a full factorial design with male strain and hermaphrodite strain as fixed factors. Subsequent posthoc tests were performed with Tukey HSD. A similar ANOVA was performed to assess the effect of different numbers of males on offspring numbers of repeatedly mated hermaphrodites. In this case, number of males was used as a fixed predictor in the model. With respect to mating behaviour, the time measurements until first contact or first copula were always terminated after 60 min, resulting in non-continuous data. Therefore, differences between crosses were assessed using the Wilcoxon sign rank test. The variation in the number of copulas during this time frame were examined with the Fisher exact test. Differences in the number of contacts or copulations over 14 observation points within a 9 h interval were assessed with the Wilcoxon sign rank test, since the data were non-parametric.All statistical analyses were done with the program JMP IN version 5.1.2  or SPSS version 14.0 . The male proportion over time in populations of the different isolates was evaluated using logistic regression based on a full factorial model with time and either population size or C. elegans isolates. Eight strains were tested at two different arbitrarily chosen population sizes (75 and 150). The proportion of males was significantly affected by the factor time, the strain studied, and also the interaction of the two. In particular, males disappeared completely from the cultures of some strains, among them N2. In contrast, in other strains, among them CB4856, the cultures appeared to reach a stable frequency of males after about two weeks  for N2 and CB4856 . The main experiment focused on the consequences of repeated mating for individual males, which is likely to be realistic in most populations, where male frequency is usually less than 0.01 . Three pP > 0.05; Figure C. elegans isolates [CB4856 males had significantly more successful copulations Figure  and signisolates .t37 = -4.65, P < 0.001). In general, our results on total cross-progeny sired by N2 males , nor in the time until first successful spicule insertions , nor in the number of replicates with successful spicule insertions . We conclude that the CB4856 males are neither generally better in finding their first mates nor in achieving their first spicule insertion. This result is consistent with the previous finding that N2 and CB4856 show similar behavioral responses to a hermaphrodite-derived cue [In a separate set of experiments we measured different aspects of mating behavior within either the first one or nine hours. As in the above experiments, we combined an individual male with 14 hermaphrodites, although in this case both were always of the same strain . Within the first one hour, the different crosses did not show any significant variation, neither regarding the time until first male-hermaphrodite contact  and spicule insertions . From these observations, we conclude that over time CB4856 males achieve a higher rate of mate contacts and spicule insertions than N2 males. These results are in excellent agreement with the results presented in Fig. However, over 14 observation points within the first 9 hours, the CB4856 × CB4856 crosses produced significantly more male-hermaphrodite contacts  could have also affected the observed differences in male persistence. We have not as yet measured this trait. However it is unlikely to have significantly influenced the results on mating efficiency in Fig. C. elegans populations, mating only once, if at all, might be realistic. However, in our male maintenance assays  and the proportion of self-progeny was small. The origin of the male did not make any significant difference . Analysis of the first two days only produced essentially identical results [see Additional file In agreement with earlier literature , we founAt first sight, this result appears to contradict our findings from the male maintenance assays, where N2 populations loose males rapidly in contrast to CB4856. However, hermaphrodites in the repeated mating experiment were exposed to "new" virgin males every day (most likely with high mating efficiency), whereas hermaphrodites in the male maintenance assay encounter the same males over consecutive days, which are likely to show reduced mating efficiency over time (due to the likely high energy demand per mating as well as sperm depletion). The reduction in mating efficiency is particularly pronounced in N2 (see results from the mating behaviour experiments above). Therefore, we expect fewer matings, a relatively larger number of self-progeny, and thus a continuous decrease of males in the N2 populations over time.A possible higher cost of multiple mating may further contribute to more rapid initial male decline in N2 in the male maintenance assays. Repeated mating with increasing numbers of males caused a significant reduction in offspring number in both strains  CB4856 males achieved a larger number of successful copulations and therefore sired more cross-progeny than N2. Consequently, a mixed-gender CB4856 population will contain a larger number of cross-fertilized hermaphrodites and thus produce more males than a corresponding N2 population. The resulting higher frequency of males should further enhance cross-fertilization rates in CB4856, because male density positively links with hermaphrodite mating rates . ii) UnmVW did all the bench work and participated in the experimental design, the analysis of the data and the writing of the manuscript. HS participated in the experimental design and the data analysis. He did all the statistical analyses and he co-wrote the manuscript together with AS. AS participated in the experimental design and the data analysis. He coordinated the whole work and supervised the practical work. He co-wrote the manuscript together with HS. The contributions of HS and AS should be considered equal. All Authors read and approved the final manuscript.Supplementary table 1. Logistic regression of male proportion in different natural isolates.Click here for fileSupplementary table 2. Male proportion averaged over days 16 to 32 for different strains and two population sizes.Click here for fileSupplementary table 3. Logistic regression of male proportion in N2 and CB4856 with different population sizes.Click here for fileSupplementary table. Male proportion on day 32 for different population sizes of the strains N2 and CB4856.Click here for fileSupplementary table 5. Variation in the number of contacts and spicule insertions within the first 9 hours.Click here for fileSupplementary table 6. Variation in the number of cross- and self-progeny per repeatedly mated hermaphrodite for the whole experimental period.Click here for fileSupplementary table 7. Variation in the number of cross- and self-progeny per repeatedly mated hermaphrodite for the first two days only.Click here for fileSupplementary table 8. Variation in total hermaphrodite offspring number after repeated mating to either 1, 3, 6, or 12 males.Click here for file


By applying cumulative sums (CUSUM), a quality control method commonly used in manufacturing, we constructed a process for detecting unusual clusters among reported laboratory isolates of disease-causing organisms. We developed a computer algorithm based on minimal adjustments to the CUSUM method, which cumulates sums of the differences between frequencies of isolates and their expected means; we used the algorithm to identify outbreaks of Salmonella Enteritidis isolates reported in 1993. By comparing these detected outbreaks with known reported outbreaks, we estimated the sensitivity, specificity, and false-positive rate of the method. Sensitivity by state in which the outbreak was reported was 0%(0/1) to 100%. Specificity was 64% to 100%, and the false-positive rate was 0 to 1.


To assess the efficacy of facet joint infiltrations for pain relief in 44 selected patients with chronic nonradicular low back pain (LBP).Forty-four patients with chronic LBP of more than 3 months' duration were selected for facet joint infiltration. The majority (n = 24) had facetal pain with no evidence of significant facetal arthropathy on imaging. Fifteen patients had radiological evidence of facetal arthropathy, one had a facet joint synovial cyst, three were post–lumbar surgery patients, and two patients had spondylolysis. Facet joint injections were carried out under fluoroscopic guidance in 39 patients and under CT guidance in 5 cases. Pain relief was assessed using the visual analog scale at 1 h post-procedure and, thereafter, at 1, 4, 12, and 24 weeks.A total of 141 facet joints were infiltrated in 44 patients over a 2-year period. There was significant pain relief in 81.8% patients 1 h after the procedure, in 86.3% after 1 week, in 93.3% after 4 weeks, in 85.7% after 12 weeks, and in 62.5% after 24 weeks. No major complications were encountered.Facet nerve block was found to be a simple, minimally invasive, and safe procedure. With meticulous patient selection, we achieved long-term success rates of over 60%. We conclude that this method represents an important alternative treatment for nonradicular back pain. Chronic low back pain (LBP) has assumed endemic proportions, with an annual prevalence of 5–20% in the industrialized world. It is no2Chronic LBP of more than 3 months' duration, not responding to conventional drugs, exercise, and physiotherapySymptoms suggestive of facetal pain; i.e., LBP with or without radiation to the buttocks, thigh, or groin; pain increasing on hyperextension; and pain when initiating movementFocal tenderness over the facet joint elicited by digital pressurePost–lumbar disc surgery patients with persistent pain and no MRI evidence of arachnoiditis or recurrent disc diseaseAll patients were initially assessed by a neurosurgeon before referral to the anesthesiologist-run pain clinic. A total of 44 patients were included in the study on the basis of the following criteria:All the patients had a preprocedure MRI for categorical exclusion of a discogenic cause for the LBP. Evidence of facetal arthropathy on MRI was noted but was not considered an inclusion criterion by itself.A neurological deficit in the lower limb or a positive sciatic nerve stretch sign Evidence of nerve root compression at the expected level on MRIClinical or imaging evidence of infection or neoplastic diseasePossible pregnancy, bleeding diathesis, or anticoagulant therapyHistory of sensitivity to local anestheticsThe exclusion criteria were:4–5 and L3–4 levels, respectively.There were 23 men and 21 women included in the study; the ages ranged from 20–74 years. The duration of symptoms varied from 3 months to 2.5 years (mean duration: 11 months). In our patient population, the largest subgroup (23/44) had clinical signs and symptoms of facetal pain but no evidence of significant facetal arthropathy on imaging (radiography / CT / MRI). Fifteen patients had clinical as well as radiological evidence of facetal arthropathy , one patThe procedure was explained to the patient in detail and written consent was obtained. Injections were performed under fluoroscopic guidance in most (39/44) patients. CT guidance was used in five cases. The levels and side(s) to be injected were selected by the treating pain physician on the basis of the tenderness elicited over the joint, correlated with imaging findings, if any.Fluoroscopic procedure: The patient was placed in the prone position with a pillow under the abdomen to correct the lumbar lordosis. The joint to be injected was located and marked. The x-ray tube was then slowly rotated till the joint appeared in profile as two parallel lines. After cleaning and draping, and administration of local anesthesia, a 22-G spinal needle was inserted in line with the x-ray beam till it contacted bone at the lip of the facet joint. With fine movements the needle tip could be made to enter the joint with a distinct ‘give.’ In the early cases, we confirmed the intra-articular position of the needle by injecting 0.5 ml iohexol  under fluoroscopy. Later, with increasing experience, we found that confirmation of correct needle placement could be made by feel and by viewing the joint in the lateral oblique projection [ojection . Once thCT-guided procedure: The patient was placed in the prone position and 5-mm axial sections were obtained at the level of interest to determine the entry site and the angle of approach. The entry site was marked on the skin and a 22-G needle was advanced into the joint [he joint . The druPain relief was assessed using a visual analog scale (VAS), with a score of 0 denoting ‘no pain’ and a score of 10 the ‘worst pain possible’ . The VAS4–5 (31.9%) followed by L3–4 (26.5%) [A total of 141 facet joints  were infiltrated in 44 patients, over a period of 2 years. The maximum number of infiltrations were at L (26.5%) . Only a The number of patients with significant postprocedure pain relief on day 1 and at 1, 4, 12, and 24 weeks is detailed in At 4 weeks, the three patients who did not respond were re-injected at the affected levels. Of these, one responded to the reinjection; the other two remained non-responders throughout the period of follow-up. Four of the patients who had responded well at 4 and 12 weeks required repeat injections between 16 and 20 weeks to maintain the pain relief. Another four patients who had good relief till 12 weeks received repeat injections at 24 weeks as the effect waned.A further analysis of the pain relief in the different subgroups of patients is shown in We saw no major complications. Five patients had minor undesired effects in the form of soreness or local skin bruising; these symptoms lasted 2–3 days and subsided without treatment.Facet joints are true synovial joints which are innervated by the medial branches of the dorsal rami. The presence of nociceptive nerve fibers in the synovium and fibrous capsule of the facet joints suggests that these joints may be a cause for LBP when they are stressed due to segmental instability, inflammatory synovitis, degenerative arthritis, or a combination of all of these.6 Based o2Unfortunately, there are no clinical or imaging findings to definitively diagnose facetal pain and very often the term ‘facet syndrome’ is used as a ‘dustbin diagnosis’ when nothing else fits. However, there are some features that are characteristic of facetal arthropathy. These include diffuse referred pain over the buttock and posterolateral thigh, exacerbation of pain with hyperextension or lateral bending, tenderness localized over one or more facet joints on deep pressure, and absence of root pain or neurological deficits.Imaging is not reliable for the diagnosis of facetal osteoarthritis since the changes seen on x-ray, CT, and MRI are equally common in patients with and without LBP, and most studies have failed to show a correlation between radiologic imaging findings and facet joint pain.–10Facet joint injection with local anesthetic and steroid is the simplest and most common procedure for facet joint–mediated pain. These infiltrations are diagnostic as well as therapeutic and the choice of guidance—whether CT or fluoroscopic—is largely a matter of personal preference and experience, as both are equally effective.910 The i9Accurate assessment of pain is a prerequisite for the effective management of patients with LBP. Of the numerous tools available to assess pain, we adopted the numerical visual analog scale (VAS) since it is an objective measure and can be used to track serial changes. Many rec12Literature describing the effectiveness of facet joint infiltrations is as abundant as it is controversial. Early studies of facetal infiltrations showed poor results and led to the conclusion that this is a nonspecific and ineffective method of treatment.15 It is 1012171012Injections for spondylolysis are a modification of facetal injections, providing good response in most patients. Some workers feel that it is the fracture site which is painful and, accordingly, infiltrate the break in the pars interarticularis. Others h1014The major complications of facet joint infiltrations are related to improper needle placement, bleeding, or infection. Complications include dural puncture, hematoma formation, spinal cord or neural trauma, spinal anesthesia, septic arthritis / spondylitis, and chemical men-ingitis. Despite We acknowledge the following limitations of our study. Firstly, we did not have a control group receiving placebo injections. Secondly, we did not carry out a preliminary diagnostic block for patient selection prior to giving the therapeutic injection. This was because we found it difficult to justify a preliminary diagnostic block followed by a subsequent therapeutic injection; such a protocol would have delayed treatment and exposed the patient repeatedly to the risk of complications and radiation exposure. Therefore we selected our patients on the basis of a good clinical examination.Recent evidence-based guidelines, published in 2007, with respect to lumbar facet joint injections have convincingly demonstrated moderate evidence for short- and medium-term (up to 6 months) relief and limited evidence for long-term relief. Facetal


Angiogenesis, the formation of new blood vessels, is a primordial process in development and its dysregulation has a central role in the pathogenesis of many diseases. Angiogenin (ANG), a peculiar member of the RNase A superfamily, is a potent inducer of angiogenesis involved in many different types of cancer, amyotrophic lateral sclerosis and also with a possible role in the innate immune defense. The evolutionary path of this family has been a highly dynamic one, where positive selection has played a strong role. In this work we used a combined gene and protein level approach to determine the main sites under diversifying selection on the primate ANG gene and analyze its structural and functional implications.We obtained evidence for positive selection in the primate ANG gene. Site specific analysis pointed out 15 sites under positive selection, most of which also exhibited drastic changes in amino acid properties. The mapping of these sites in the ANG 3D-structure described five clusters, four of which were located in functional regions: two in the active site region, one in the nucleolar location signal and one in the cell-binding site. Eight of the 15 sites under selection in the primate ANG gene were highly or moderately conserved in the RNase A family, suggesting a directed event and not a simple consequence of local structural or functional permissiveness. Moreover, 11 sites were exposed to the surface of the protein indicating that they may influence the interactions performed by ANG.Using a maximum likelihood gene level analysis we identified 15 sites under positive selection in the primate ANG genes, that were further corroborated through a protein level analysis of radical changes in amino acid properties. These sites mapped onto the main functional regions of the ANG protein. The fact that evidence for positive selection is present in all ANG regions required for angiogenesis may be a good indication that angiogenesis is the process under selection. However, other possibilities to be considered arise from the possible involvement of ANG in innate immunity and the potential influence or co-evolution with its interacting proteins and ligands. The complex network of interactions between pro- and anti-angiogenic regulators dictates that any imbalance in this process can lead to disease. Indeed, angiogenesis plays a central role in the pathophysiology of cancer, rheumatoid arthritis, diabetic retinopathy and several heart diseases (reviewed in ). Angiogicrobial  and antiicrobial  effects,icrobial , constiticrobial .4-106 lower and its enzymatic specificity is also different [The human ANG gene comprises a single exon flanked by small UTRs and codes for a 14 kDa polypeptide. The protein is synthesized with a 24 amino acid signal peptide that is cleaved to produce a mature form with 123 amino acids. Sequence analysis revealed its homology to the Ribonuclease A (RNase A) superfamily, where it was included and classified as RNase 5 ,16. ANG ifferent ; (2) theifferent ,19, probifferent . All of ifferent -23.The RNase superfamily has a highly dynamic evolutionary history, in which ANG occupies an important position. The fact that only ANG/RNase 5-like ribonucleases are found in non-mammalian vertebrates has led to the hypothesis that the RNase ancestral was structurally similar to ANG. This ancestral enzyme was most likely involved in host-pathogen interactions and did not possess an angiogenic activity . A gene Pygathrix nemaeus (Asian Douc Langur), in which the ANG gene appears to have pseudogenized [Diversifying (positive) selection had a strong influence in the RNase A superfamily evolutionary pathway: eosinophil RNases, EDN and ECP, are among the most rapidly evolving coding sequences in primates , as are ogenized .In this work we assessed the impacts of positive selection on the primate ANG gene using: (1) a gene level evaluation of the non-synonymous/synonymous ratio (dN/dS) and (2) a protein level evaluation of radical changes in amino acid properties. Several sites under positive selection were detected in the different functional regions of ANG and the effects in its structure and function have been analyzed.Miopithecus talapoin. Overall, tree topologies were mostly coherent with the accepted phylogeny of primates. The small differences detected are not surprising since the gene tree does not necessarily reflect the species tree [Neighbor-Joining (NJ), maximum-likelihood (ML) and Bayesian (BAY) tree reconstructions of the primate ANG coding sequences table  presenteies tree .Papio hamadryas, Macaca mulatta and Cercopithecus aethiops , base (B0-Bn) and ribose (R0-Rn) moieties of each RNA nucleotide  B1, where the 3' base binds and (3) B2 where interaction with the 5' base occurs. Structural comparisons between RNase A and ANG have allowed the mapping and characterization of the different catalytic subsites [1 subsite  appears to be well conserved, the most striking structural differences residing in the B1 subsite that appears to be blocked by Gln117 and partly by Phe120.RNase' A active sites can be divided in different subsites corresponding to the binding sites of the phosphate (Piewed in ). The mosubsites ,42. Only1, B1 or B2 catalytic subsites. Cluster-1 of positive selected sites under positive selection was located in the subsite P2 region, and included type I site, 4 (Ser) and three type II sites: 7 (Thr), 8 (His) and 11 (Thr). These sites were all located within the first ANG α-helix, neighboring several conserved important amino acids. Site 4 presented the greatest diversity of drastic changes in chemical and structural properties, linked to polarity, hydrophobicity, bulkiness and structural conformation. A more reduced diversity was obtained for site 7 mostly reflecting chemical changes, whereas sites 8 and 11 had few and mostly conformational properties under positive selection.As expected due to functional constraints, no evidence for positive selection was detected in amino acids with important roles to the PColobus guereza and Macaca mulatta  and 84 (His), type II sites 82 (Lys) and 93 (Gln), located close or within the anti-parallel β-sheet formed by β-strands B1, B4 and B5. Sites 41 and 82 lie at the C-terminal region of the active site, while sites 84 and 93 are located further up, facing the exterior of the protein. Site 41, neighboring two important active site residues Lys40 [P2 and B1, shaping the local chemical and conformational environment without significantly interfering with the position critical residues, which are probably subject to purifying selection.Overall, the results for the two clusters located in the active site region suggest that selective forces act in several of the more permissive sites of subsites P31RRRGL35 [The nucleolar import of ANG was mapped in the position 1RRRGL35 , a regio1RRRGL35 .Cluster-3 of sites under positive selection is located in this region with a type I site 34 (Gly) and a type II site 32 (Arg). Analysis of drastic changes in amino acid properties for site 32 resulted in a total of five properties indicative of changes in polarity and helical conformation. Site 34 had an elevated number of drastic changes in properties as a result of the broad diversity of amino acid substitutions  and Arg66 and a type II site: Val103. For the first of these sites, a total of nine properties were subjected to drastic changes: four chemical properties, four structural properties and one uncategorized property. These are mostly related to polarity and volume/spatial occupation. Site 66 presented the highest number of properties subject to drastic changes in this analysis with a total of 16 out of 31 properties – nine chemical and seven structural. Overall, the evolutionary changes observed for sites 63 and 66 influence the interactions performed by this region. However, further information about the interactions between this region of ANG and other proteins would be required in order to further evaluate the effects of positive selection in this region.Site 103  is a buried residue, but was also included in this cluster, since its α-carbon is relatively close to sites 63 and 66 . This site is part of the hydrophobic core of the enzyme  and 52 (Ser), located in helix 3 outside of the main functional regions. The support for site 52 as being under selection was weak, only obtained with the NJ topology in PAML. As for site 51 no particular functional or structural information was available, besides being a rather flexible residue table  that is Zhang and Rosenberg [Previous work by osenberg  had repoEurtherian ancestral already had a deeply invasive placenta and that the different forms of placentation currently observed were the result of clade-specific or convergent evolution.Two events are generally used as reference for physiologic and pathologic angiogenesis: placentation and cancer. All the primate groups analyzed in this study have hemochorial placentas, the most invasive form of placentation, where a direct contact between the placental and maternal circulations is established. Expression studies are only available for human placentas and indicate significant increases in the ANG levels in last trimester placentas, correlating well with the dramatic increase in placental vessel density and in fetal growth that occurs in this period . FurtherAs for cancer, the overexpression of ANG has been documented in many tumors and appears to correlate well with disease establishment, progression and in some cases aggressiveness (reviewed in ). Many aZhang and Rosenberg [Crespi and Summers in relation to cancer [It is noteworthy that invasive placentation and cancer progression share many features, like the invasive cell phenotype, vessel reorganization and neovascularization at the site of implantation. Therefore, any evolutionary changes that impact placentation are likely to have implications in cancer. Previously, osenberg  suggesteosenberg , and sugo cancer . These aThe association between ANG mutations and disease has only recently been observed in amyotrophic lateral sclerosis (ALS) patients , mostly in vitro [in vivo [Although angiogenesis appears to take centre stage as a driving force for positive selection in the ANG gene, it is still possible that there are other processes contributing to selective pressures. Several lines of evidence have accumulated supporting the existence of other ANG functions: (i) the expression of ANG mRNA is low in the developing fetus, reaching maximum levels in the adult, a pattern not temporarily related to vessel development  and has in vitro ,12, but in vitro , whose s[in vivo . Furthera priori modulate the interactions with other proteins or ligands. It is also noteworthy that some of the sites detected under positive selection in this study were found to be involved in contacts with the ribonuclease inhibitor (RI) in the crystal structure of the ANG-RI complex  using GETAREA figure . This prex table . FurtherIn this work we aimed at evaluating in detail the amino acid sites under positive selection in the primate ANG gene, including its possible structural and functional impacts. Using a maximum-likelihood gene level analysis we obtained evidence for positive selection on the ANG gene. Posterior site specific analyses allowed the identification of 15 sites with strong evidence of positive selection, further corroborated by a protein level analysis that showed that five of these sites also had an elevated number of amino acid properties suffering radical changes. The mapping of these sites in the ANG 3D-structure revealed five clusters in specific regions within the main functional regions of the protein. In the active site region, positive selection appears to modulate the chemical and structural characteristics of a few permissive sites without affecting the position of the critical residues. Significant chemical and structural divergence is further observed in two other regions, the nucleolar location signal and 'cell-binding' site, clearly having a potential to influence the interactions performed through them.Given that evidence of positive selection was detected in all ANG functional regions required for angiogenesis, it is tempting to indicate angiogenesis as the process underlying selection. It is unclear, however, if it is physiologic or pathologic involvement of ANG in angiogenesis that dictates the selective pressures. The two prototypical situations, placentation and cancer, share common features in the primate species analyzed and the hypothesis of selective pressures motivated by antagonistic coevolution appears to set a common ground explanation on how the evolutionary changes motivated by physiologic processes involving ANG can lead to an increased risk of cancer. However, other processes may also influence ANG's adaptative evolution, and the possible involvement in innate immunity is particularly interesting since the host-pathogen 'arms-race' is a common origin for selective pressure. Also, one must consider the possibility of ANG/ligands co-evolution given that most sites under selection are exposed in the surface of the protein and can mediate interactions. Future experimental characterizations of ANG function, physiologic mechanism and interactions will allow further evaluation of these hypotheses.Platyrrhini (new world monkeys) and Catarrhini (old world monkeys and hominids). Reference sequences were available for Homo sapiens and Pan troglodytes, the remaining primate sequences had been previously published [ANG coding sequences were retrieved from GenBank for 14 different species representative of the two main branches of the simian primates: ublished ,31  and a more general, alternative model that admits positive selection (ω > 1). The one ratio model (M0) and three pairs of site specific models were used, as suggested in the PAML user's guide: M1a  versus M2a (PositiveSelection); M7 (Beta) versus M8 (Beta&ω) and M8a (Beta&ωs = 1) versus M8 (Beta&ω). The significance of the LRT between the neutral and alternative model was assessed as described before and due to possible complications with non-estimable parameters, the following degrees of freedom were used, as they are expected to be conservative [Alignments and the NJ/ML/BY trees were used for posterior molecular evolution analyses. Evidence for positive selection on ANG was first evaluated using likelihood ratio tests using the CODEML algorithm of the PAML 3.14b package . We testervative : M1a vs.A protein level analysis was performed using TreeSAAP 3.2 . This prHomo sapiens sequence as query) were submitted to the server. The 2ANG pdb file was used as reference and the phylogenetic tree was the one generated by ConSurf. Confidence intervals for the conservation scores estimations are calculated and when the number of sequences is small, the confidence interval tends to be large. Amino acid positions that are assigned confidence intervals that are too large are considered to be below the confidence cut-off. This was the case for sites 32, 34 and 52 in the primate ANG alignment.The conservation of the sites detected under selection in the RNase A family was tested using the ConSurf web server . This prThe main functional regions and sites under positive selection were mapped on the X-ray crystallographic structure of Human ANG, retrieved from the Protein Databank as 2ANG . VisualiThe surface exposure of amino acids was assessed using GETAREA 1.1  web inteECP – Eosinophil cationic proteinEDN – Eosinophil derived neurotoxinLRT – Likelihood ratio testUTR – Untranslated regionThe author(s) declare that there are no competing interests.DSO performed all phylogenetic, evolutionary and structure-function analyses and drafted the manuscript, AA participated in the genetic analyses, design, drafting and coordination of the study, MJR participated in the drafting and coordination of the study. All authors read and approved the final manuscript.Neighbor-Joining phylogenetic tree of the primate species analyzed showing branch labeling as used in the PAML branch analyses.Click here for fileLikelihood ratio tests for PAML branch models.Click here for fileCategorization of TreeSAAP properties. TreeSAAP properties divided in three categories based on their nature: chemical, structural or others.Click here for file


Plasticization effects are also observed with higher hydrocarbons such as propylene, propane, aromatics or sulfur containing aromatics. Unfortunately, these components are present in mixtures of high commercial relevance and can be separated economically by single membrane units or hybrid processes where conventional separation units are combined with membrane-based processes. In this paper the advantages of carboxy group containing 6FDA  -copolyimides are discussed based on the experimental results for non cross-linked, ionically and covalently cross-linked membrane materials with respect to the separation of olefins/paraffins, e.g. propylene/propane, aromatic/aliphatic separation e.g. benzene/cyclohexane as well as high pressure gas separations, e.g. CO2/CH4 mixtures. In addition, opportunities for implementing the membrane units in conventional separation processes are discussed.Functionalized copolyimides continue to attract much attention as membrane materials because they can fulfill the demands for industrial applications. Thus not only good separation characteristics but also high temperature stability and chemical resistance are required. Furthermore, it is very important that membrane materials are resistant to plasticization since it has been shown that this phenomenon leads to a significant increase in permeability with a dramatic loss in selectivity. Plasticization effects occur with most polymer membranes at high CO Over 50% of energy costs in the chemical industry are used for the separation of gaseous or liquid mixtures . SeparatMembrane devices offer new opportunities for the separation of gaseous or liquid mixtures. Compared to conventional distillation units, membrane devices are much smaller and processes can be conducted at lower temperatures. In certain cases, the combination of distillation and membrane units, so called hybrid processes, have been established. For the separation of olefin/paraffin mixtures it has been estimated that membrane based hybrid processes could save approximately 40–50% of the production costs . The worIn general, membranes are very thin layers, which can have different structures. They are divided into porous membranes and solution-diffusion membranes. Porous membranes are well established in typical filtration processes e.g. micro-, ultra-, and nanofiltration. The particles to be separated have diameters between 10 and 1000 nanometers and will be held back from the membrane due to the fact that the pores of the membrane are smaller than the particle size.If the size of the components to be separated is less than 1 nanometer – which is the case for many gaseous, vaporous and also liquid components that have to be removed from process streams – then mainly so called solution-diffusion membranes are used. This type of membrane does not have pores but free volume sites which exist due to restricted motion and packing density of the polymer chains.The mass transport through solution-diffusion membranes can be described with the solution-diffusion model . Based oIn order to optimize material properties for the solution-diffusion process through the polymer matrix, approaches can be made taking the molecular structure into account .Thus, it is supposed that a certain free volume exists in polymers. This is the volume, which cannot be occupied by polymer chains due to conformational constraints. Within this free volume, transient gaps are formed which can accommodate, e.g., gas molecules. According to the driving force, the components have to be transported by successive movement between transient gaps close to the feed side to those close to the permeate side. The movement necessary for the transport of the components between the microvoids is possible due to thermal motion of segments of the polymer chains.nA), normalized by the transmembrane partial pressure (ΔpA) and thickness (ℓ), the permeability (PA) is defined, as shown in Polymeric membrane materials are generally characterized by the transport properties permeability and selectivity. Permeability is a measure of the productivity of the membrane and selectivity is a measure of the separation efficiency. For polymer films without any support, which are used as membrane materials in this review, the flux  between A and B is defined as the ratio of their permeabilities  together with p-toluenesulfonic acid as a catalyst to the casting solution. After the solvent has been evaporated at 70 °C, the membranes obtained are stored for another 24 h at 150 °C at 80 mbar in order to carry out the cross-linking reaction.Covalent cross-linked membranes were prepared by dissolving the carboxy group containing copolyimide in dry Ionically cross-linked membranes have been prepared by dissolving the carboxy group containing copolyimide in tetrahydrofuran (THF) and adding a stoichiometric amount of aluminium acetylacetonate or zircon(IV) acetylacetonate to the casting solution. After evaporation of the solvent, the membranes are dried in a vacuum oven at 150 °C and 80 mbar in order to perform the cross-linking reaction.In the following sections it will be shown which cross-linking method, ionic or covalent, will be the most effective to reduce undesired plasticization effects occurring in different separations. Furthermore, different ionic cross-linkers are compared, since it was expected that higher cation loadings would lead to more effective cross-linking.According to 2 content, are discussed in detail.Several membrane-based separations are already well established commercially. However, the number of new potential applications is steadily increasing –6. ThereThe separation of olefin/paraffin mixtures is rather difficult because of the small differences in physical properties, e.g., boiling points. Currently, such separations are carried out by energy intensive low temperature distillation. Huge splitter columns are necessary to separate the mixtures of saturated and unsaturated hydrocarbons to obtain, e.g., propylene of sufficient purity for polymerization reactions. As shown in However, membrane based processes for olefin/paraffin separations are currently not possible because suitable membrane materials are not commercially available. It has been found that polymeric membranes, e.g., silicone rubber, polysulfone, cellulose acetate, PDMS, 1,2-polybutadiene and polyethylene are not suitable for this kind of separation because the separation factors are far too low –46. MuchUnfortunately, polyimides are sensitive to plasticization due to higher hydrocarbons. It has been previously shown that neither ethane nor ethylene plasticize polyimide membranes but propane, propylene and higher hydrocarbon do ,45,48–49As reference material, the 6FDA-4MPD was used as non cross-linkable membrane. In order to compare the propylene/propane separation characteristics of a 6FDA-4MPD polyimide membrane with a cross-linked copolyimide membrane, a copolyimide was synthesized substituting 20% of the 4MPD by the DABA diamine. The copolyimide obtained was the 6FDA-4MPD/6FDA-DABA 4:1. The structure is shown in The separation of aromatics/aliphatics is receiving more and more attention, as the benzene content in gasoline is, by law in Europe, limited to less than 1%. Discussions on the reduction of toluene and polynuclear aromatic compounds in gasoline are ongoing. Although the feed concentration for the aromatics in reformate streams is usually between 40% and 50%, it sometimes happens that much higher concentrations occur over a short time period. Therefore it is important to ensure that the membrane material is stable and additionally, that the separation characteristics – especially the selectivity – will not change drastically. It has been demonstrated that polyimides in principle are suitable for the separation of aromatic/aliphatic mixtures but unless they are cross-linked, the stability is poor especially at high aromatic concentrations and elevated feed temperatures. In order to circumvent these problems in practical applications, different copolyimides have been synthesized and their stabilities after exposure to high benzene concentrations tested. Pre-treatment of the membrane material prior to its use is referred to as conditioning. The effects of varying the composition of the conditioning agent were also investigated in this work. 6FDA-4MPD/6FDA-DABA 4:1 was used as the basic membrane material and non cross-linked membranes were prepared and conditioned in pure toluene and in a mixture of 90:10 toluene/cyclohexane, respectively. The results of the pervaporation experiments at 60 °C and at a permeate pressure of 20–25 mbar are shown in It can be seen clearly that the membrane conditioned in pure toluene shows a much higher flux than the one conditioned in 90:10 toluene/cyclohexane. This effect is even more pronounced at higher toluene concentrations in feed. Remarkably, there is no significant difference in the selectivity for the different conditioned membranes within experimental error. From our experience in pervaporation experiments, the error range for the selectivity is ±10%, whereas for the flux it could be as much as 15%, depending on the homogeneity of the membranes. However, from the experiments performed, it can be concluded, that with appropriate conditioning, fluxes can be increased without any loss in selectivity.Further experiments have been carried out in order to investigate the separation characteristics for covalently cross-linked copolyimide membranes compared to ionically cross-linked copolyimide membranes and non cross-linked membranes prepared from the same type of polymer. The polymer 6FDA-4MPD/6FDA-DABA 4:1 copolyimide was used and the covalent cross-linker was 1,4-butanediol. An ionically cross-linked membrane was prepared from the basic polymer material by adding 10% of the stoichiometric amount of cross-linker (based on the free carboxy groups of the polymer). For the ionic cross-linking, zircon(IV) acetylacetonate was used since it was expected to be more efficient than aluminium acetylacetonate because of the higher charge of the cation formed during the cross-linking reaction. The mixture to be separated consisted of toluene and cyclohexane and the experiments were carried out at 60 °C at a permeate pressure of 20–25 mbar.The results obtained  show thaFrom the experiments it can be concluded that conditioning of the membrane material is favourable since the flux can be increased without a loss in selectivity. It can be also seen that cross-linking is not necessary for the aromatic/aliphatic separation at moderate temperatures, e.g. 60 °C, but it presumably will increase the life time of the membrane with respect to the proposed hybrid process shown in 2 is introduced into the oil field, especially if the oil is distributed in porous layers. As shown in 2S and a small amount of water. However, it is important to hold back the CO2 present in this stream because it is well known that CO2 is one of various compounds responsible for the greenhouse effect. 2 is removed through the membrane and can then be re-used after compression. With this process not only could the emission of CO2 be drastically reduced but also the natural gas can be recovered in this case instead of burning it which is generally carried out if the quantity and quality are too low.In the area of natural gas treatment a number of different applications are of great interest. In tertiary oil production supercritical CO2 content and the high pressure of the mixture. For economic reasons more and more membrane based processes in natural gas treatments are operated with polyimides as the membrane material instead of cellulose derivatives, since the intrinsic transport properties for the polyimides are much better. However, strong plasticization effects occur with non cross-linked polyimides generally at 10–20 bar partial CO2 pressure in the feed. Therefore, in this context cross-linkable copolyimides are of interest because they offer plasticization resistance up to much higher CO2 pressures and, in addition, they have better chemical resistance. In Another very interesting application is the treatment of natural gas in offshore deposits. So far a huge number of gas resources are known worldwide, which cannot be exploited because of the high CO2 pressure of approximately 15 bar. The plasticization is indicated by a large increase in permeability caused by increasing segmental motion of the polymer chains. 2 pressures whereas the covalently cross-linked membrane is resistant to plasticization up to a CO2 pressure of approximately 30 bar. Similar effects have been found for different copolyimide structures [It can be seen that the non cross-linkable reference polyimide, 6FDA-4MPD indeed starts plasticizing at a pure COructures .2. Another plausible reason might be that the CO2 with its strong solvation effect is able to weaken the ionic interactions in the ionic regions.It is assumed that ionic cross-linking leads to a much lower plasticization resistance compared to covalent cross-linking because ionic aggregates are formed due to electrostatic interactions, in this case between aluminium cations and carboxylate anions. Thus, heterogeneous regions with ionic and non-ionic domains are produced. The non-ionic regions consist mainly of polymer chains which are sensitive to plasticization in the presence of strong plasticization agents such as CO2 permeabilities and ideal CO2/CH4 selectivities, which are the ratio of pure gas permeabilities, are reported for experiments performed at 10 bar feed pressure and 35 °C. Compared to the reference polyimide 6FDA-4MPD, the modified but non cross-linked 6FDA-4MPD/6FDA-DABA 4:1 copolyimide shows a much lower permeability but higher selectivity. The lower permeability results because the DABA in the polymer structure provides a much lower free volume than the 4MPD. With both cross-linked versions of the 6FDA-4MPD/6FDA-DABA 4:1, the permeability can be increased whereas the selectivity is not changed significantly compared to the non cross-linked 4:1 copolyimide. However, the ideal selectivities obtained with the different cross-linked 6FDA-4MPD/6FDA-DABA 4:1 copolyimides are not high enough to be commercially attractive. It should be noted that these are ideal selectivities and often the selectivities obtained with feed mixtures are even lower due to the fact that the diffusivity of the individual components are influenced by each other. This leads to effects where the slower permeating component is accelerated whereas the faster permeating component is slowed down.In 3 groups, which being bulky groups cause restricted rotation around the main polymer chain and also provide a high free volume.In order to increase the selectivity the basic polymer structure was slightly modified. Thus, part of the 4MPD was substituted by the 6FpDA which is well known for both its high selectivity and high permeability due to the –CF2/CH4 feed mixture at 35 °C are presented for the 6FDA-6FpDA/6FDA-4MPD/6FDA-DABA 3:1:1 cross-linked covalently with ethylene glycol and the ionically cross-linked with aluminium acetylacetonate. The results mirrored those found for the 4:1 copolyimide. The ionically cross-linked structure began to plasticize very early , whereas the ethylene glycol cross-linked structure shows nearly constant permeability and a selectivity which is approximately 20% higher than the selectivity achieved with the ionically cross-linked structure. From the separation diagram it is also obvious that the selectivity is dependent on the feed pressure. With increasing feed pressure the usual decrease in selectivity was found, which is due to the interactions between the single components in the feed mixture as previously discussed. However, the modified polymer structure shows good selectivity for CO2 over methane between 30 and 40, which indeed is attractive for commercial applications because it is much higher than the separation factors of the cellulose derivatives in current use.In 2/CH4 as well as propylene/propane mixtures that the prepared cross-linked copolyimide membranes are much more plasticization resistant than non cross-linkable polyimide membranes used as reference substances. In the separation of aromatic/aliphatic mixtures, it was found that with conditioning, high fluxes combined with high selectivities can be achieved, e.g., with a 50:50 toluene/cyclohexane mixture a toluene concentration in permeate of approximately 85% can be reached. In the propylene/propane separation a 50:50 feed mixture could be concentrated to more than 90% propylene in permeate with a covalently cross-linked copolyimide structure of 6FDA-4MPD/6FDA-DABA 4:1. It was found that in the pressure range investigated (up to 4.2 bar) no plasticization was found with the cross-linked structures, whereas for the non cross-linked structure plasticization starts at a very low feed pressure of around 2 bar.It has been shown for the separation of high-pressure mixtures of CO2 from natural gas, experiments with different cross-linked membranes showed that the covalently cross-linked copolyimide structure shows a much higher plasticization resistance and better selectivity in mixed gas experiments compared to the ionically cross-linked structures.For the removal of CO