Recent studies investigating the health effects of air pollution have proven an existing impact around and below international air quality guidelines and standards. These studies were based on accessible data from official registers managed by public authorities. The protocol followed in BAPHE project is described; its benefits and disadvantages are presented and discussed in this paper.Based on the review of several international studies we developed a custom made approach in BAPHE  project in order to analyze the short term health effects of air pollution taking into consideration the lack of data availability from official sources.2.5 and PM10 concentrations were measured in Beirut for the period starting from the 1st of January 2012 to the 31st of December 2012. The annual average concentrations of PM10 and PM2.5 exceeded WHO’s annual average limits by 150 % and 200 %, respectively. Health data for 11,567 individuals were collected over 12 months. A variation of hospital admission causes was observed by age categories and gender.PMThis article presents a simple protocol and the descriptive results of its application in the frame of an eco-epidemiological study in Lebanon. We believe that this work is not only important on a local scale, but it could be helpful for environmental epidemiological studies in other countries. In some countries, epidemiological studies have been very well developed and investigators highlighted the effects of moderate and low levels of air pollution on health; trying to generate evidence of interactions between pollutants and local or regional conditions –8. Howev3 of daily PM10 levels and 5 μg/m3 for PM2.5 levels ) per 10 μg.m−3 rise in daily mean pollutant concentration for PM10 and 1.016 [95 % CI 1.000–1.032] for PM2.5 on the same day. A nearly significant association was found between particles and total circulatory admissions for adults and elderly groups in the same day. The results of the regression model were subject to a detailed article [The application of an autoregressive Poisson model was used to evaluate the association between daily concentrations of particulate matter and respiratory and cardiovascular emergency hospital admissions after controlling for confounders. All variables were measured during 1 year from January 2012 to December 2012. Relative risks of admissions for respiratory and cardiovascular diseases were calculated for an increase of 10 μg.m article .To extract information from emergency hospitals’ registers we trained individuals in the 7 participating hospitals to collect accurate data. Several causes of emergency admissions can be considered, an example of diagnosis category and subcategory is described in Table The total number of case extracted from ED registers are remarkable knowing that it is the first time in Lebanon that an environmental epidemiological study concerns such large number of individuals as BAPHE. Other obtained results are quiet impressive, especially the number of exceeding days.If we compare our results to a population based study in Ontario Canada, the sex differences in hospital admissions from emergency departments in asthmatic adults, showed that women were more likely to be admitted than men in ED. Following , the higHowever, our study presents some weaknesses. The disadvantage of studies like BAPHE involves in data collection since the health system in Lebanese hospital is not yet computerized. The hard part of the project was to collect the data manually. On another hand, this operation allows an efficient control of data through database checking by 3 different qualified experts.If we consider the fact that time series studies have lack of individual measurements in exposure and outcomes and the possible presence of unmeasured confounders, this could be a weak point for our study. However, in BAPHE as in international studies we worked on the short term effects by using long series of small units (days) to minimize errors , 27. In For decades, the effects of air pollution on human health were subject of many researches in public health in European, US and Asia cities. Several techniques and methods of analysis have been developed. Thus, this article presents a simple protocol and the descriptive results of its application in the frame of an eco-epidemiological study in Lebanon. We believe that this work is not only important on a local scale, but it could be helpful for environmental epidemiological studies in other countries. We have indicated how to collect reliable data on air pollution and health outcomes in order to investigate health impact of air pollution at urban level.Therefore, BAPHE will provide a unique opportunity to assess the relation between high levels of air pollution and health effects in a country like Lebanon.Finally, our results will contribute to set common targets and objectives for air quality and public health, guidelines to prevent, reduce or mitigate the health effects of air pollution in the city of Beirut.


Over the past 30–40 years, various carbon implant materials have become more interesting, because they are well accepted by the biological environment. The traditional carbon-based polymers give rise to many complications. The polymer complication may be eliminated through carbon fibres bound by pyrocarbon (carbon/carbon). The aim of this study is to present the long-term clinical results of carbon/carbon implants, and the results of the scanning electron microscope and energy dispersive spectrometer investigation of an implant retrieved from the human body after 8 years.Mandibular reconstruction (8–10 years ago) was performed with pure (99.99 %) carbon implants in 16 patients . The long-term effect of the human body on the carbon/carbon implant was investigated by comparing the structure, the surface morphology and the composition of an implant retrieved after 8 years to a sterilized, but not implanted one.Of the 16 patients, the implants had to be removed earlier in 5 patients because of the defect that arose on the oral mucosa above the carbon plates. During the long-term follow-up, plate fracture, loosening of the screws, infection or inflammations around the carbon/carbon implants were not observed. The thickness of the carbon fibres constituting the implants did not change during the 8-year period, the surface of the implant retrieved was covered with a thin surface layer not present on the unimplanted implant. The composition of this layer is identical to the composition of the underlying carbon fibres. Residual soft tissue penetrating the bulk material between the carbon fibre bunches was found on the retrieved implant indicating the importance of the surface morphology in tissue growth and adhering implants.The surface morphology and the structure were not changed after 8 years. The two main components of the implant retrieved from the human body are still carbon and oxygen, but the amount of oxygen is 3–4 times higher than on the surface of the reference implant, which can be attributed to the oxidative effect of the human body, consequently in the integration and biocompatibility of the implant. The clinical conclusion is that if the soft part cover is appropriate, the carbon implants are cosmetically and functionally more suitable than titanium plates. During recent years, various carbon implant materials have become of considerable interest in view of the fact that they are well tolerated by the biological environment . The traThe mechanical properties of C/C composites are very close to those of human bones . This isDuring the manufacturing of C/C composites, layers of carbon fibre fabric are combined together with a carbonaceous material to give the composite the desired shape. After multiple heat treatments and densification processes, a lightweight, yet mechanically stable composite is produced, which is almost entirely composed of carbon. The bonding and densification can be achieved with the aid of different processes and precursor materials . Such prFrom a maxillofacial surgical aspect, relatively few C/C modules are available –15. RussDuring the period between 2002 and 2005, we had the possibility to make use of C/C material for purposes of jawbone replacement, and the more than 10 years that has subsequently elapsed is sufficient for us to draw long-term consequences.What is the functional and aesthetic result? Did the implantation achieve the aim?What are the effects of the implant on its immediate and more distant environments? What is the effect of the organism on the implant? How and to what extent can the implant be damaged by the loading imposed on it because of its function and by the aggressive action of the organism?A number of questions arise when the effectiveness of medical implants is evaluated. Perhaps the most important of these are as follows:The aim of the present paper is to provide answers to these questions and to present the possibilities of the long-term application of certain carbon fibre-reinforced carbon (C/C) composites in maxillofacial surgery.Between 2002 and 2005, implants made from Carbulat™ were utilized for purposes of jawbone replacement in 16 patients  (Table In the majority of the cases (10 patients), an extensive mandibular tumour had been removed, but for various reasons ‘living bone’ could not be used for replacement primarily. The excluding factors included the previous intensive cytostatic treatment (Ewing’s sarcoma in the mandible), the rejection of the previous replacement with fibula or the fact that the tumour had been so extensive that it was removed purely for palliative reasons (e.g. a very large osteosarcoma). In 4 cases, C/C granulate was utilized for filling after the removal of a mandibular cyst, and in 2 cases, a Carbulat™ plate was used for mandibular augmentation.A granulate measuring 500–1000 μm (for the filling of cystic cavities)Compact implants with volumes corresponding to the body of the mandible (approximately 15 mm thick)  to the external surface of the mandible and which follows the curvature of the mandible , a 1.8-mm-thick titanium plate suitable for osteosynthesis was screwed to the external side of the mandible. This plate ensured that the mandible remained in its original position after the resection. This was followed (with the plate left in place) by removal of the diseased bone part. The pre-prepared Carbulat™ piece was next fitted closely at the site of the resected bone and screwed to the titanium plate. The correction was performed with a diamond cutter under physiological saline solution in a vessel suitable for this purpose, so that the black powder produced during the cutting should not contaminate the environment. In the case of the Carbulat™ net, the titanium plate could be removed in most cases if the implant could be fitted securely with screws to the distal and proximal bone parts. The most important task after the insertion of the Carbulat™ was to enclose the implant from the directions of both the face and the oral cavity with the thickest possible layer of soft parts. In the cases of patients who had undergone surgery and irradiation on a number of occasions, strain-free closure generally involved difficulty, and therefore demanded particular care.Compact Carbulat™ was applied in 2 patients, and mandibular bone was supplemented with a Carbulat™ net in 8 cases. The Carbulat™ net was used for augmentation purposes in 2 cases. In 1 patient, the facial asymmetry was corrected with a 5 × 2.5 cm net 5 years after the exstirpation of a tumour affecting the mandible. In the other case , rib transplantation had already been performed on two occasions in order to supplement the ascending branch of the mandible. Then, 10 years later, when the patient was 18 years old, the bone and soft part deficiency causing the persisting facial asymmetry was corrected with a Carbulat™ net prepared in appropriate size. This was necessary as the patient did not agree to undergo further autologous bone transplantation Fig. .Fig. 3ThFor study of the long-term effects of the interaction between an implant and the human body, the surface morphology and the average surface composition of an implanted Carbulat™ implant (retrieved after 8 years) and a sterilized but not implanted Carbulat™ reference implant were compared. After 8 years, the alloplastic material was removed from the above 18-year-old asymptomatic patient, and the composite implant was replaced with autologous bone tissue from the iliac crest.Before the measurements, the implants were cut into smaller pieces with a saw that had been cleaned with ethanol, and the pieces were dried in the air. All the tools used to mount the samples on sample holders were cleaned with an ethanol ultrasonic bath.Unimplanted (reference) implants which were examined immediately after being unpacked from their sterile packing.Pieces of the implant retrieved from the human body after 8 years. Some of these were cleaned to remove the residual soft tissue before investigations of the surface morphology and composition. This cleaning process was carried out with Enzyrim-Oss  dissolved in tap-water at a concentration of 20 ml/l, this material being used to remove soft tissues from hard tissues in forensic science .Other pieces of the implant retrieved from human body after 8 years were investigated without the enzymatic treatment in order to verify that the enzyme solution only removes the residual soft tissues from the surface and does not alter the structure or the surface composition of the C/C composite.Three types of samples were investigated:The surface morphology of the implants was investigated with a Philips XL30 scanning electron microscope (SEM), and a Bruker QUANTAX 200 energy dispersive spectrometer (EDS measurements) with an XFlash 5010 detector attached to an FEI Inspect S50 SEM was used for the measurement of the average surface composition.The surface morphology of the samples was observed with the aid of secondary electron images of the surface. In the secondary electron mode, the surface is scanned with a high-energy electron beam (in our case 20 keV) and the intensity of the low-energy (<50 eV) electrons leaving the surface is measured. The resulting images have topographical contrast, and the morphology of the surface can therefore be studied.EDS measurements were carried out to obtain quantitative information on the surface composition. During these measurements, the spectrum of the characteristic X-rays induced by the high-energy electron beam is detected. The acceleration voltage applied in our EDS measurements (20 kV) gives an informal depth of about 4–5 μm in pure carbon . The speBesides the EDS measurements, back-scattered electron (BSE) images were also taken of the surface of the samples. In these images, areas with higher average atomic numbers appear brighter than those with lower ones.Of the 4 mandibular cyst fillings, 2 were successful. In the 2 successful cases, healing was ensured by the good closure of the mucosa. In both of the 2 cases that failed , the wound opened after a few days and it was therefore necessary to remove the implanted material.Both augmentation cases proved successful. It was possible to cover the implants with a thick layer of soft parts (skin and muscle), and there was therefore no obstacle to healing.It was necessary to remove the implant from 3 of the 10 tumourous patients, in each of the 3 cases because of intraoral wound opening. The patients had previously undergone surgery and radiation treatment on a number of occasions. The intraoral suture was strained, and as a consequence of the previous events, the mucosal blood supply had deteriorated.In those cases when the Carbulat™ plate had to be removed because of tumour recurrence or in a planned manner because of the later autologous bone transplantation, it was seen that the Carbulat™ had integrated extremely well into the tissues Fig. . It was Of the 16 implants, therefore, 5 had to be removed before time because of the intraoral wound opening. Two implants were removed in order to carry out a planned bone implantation or in consequence of tumour recurrence. Three patients died between 3 and 5 years post-implantation with the implant still in place. Six implants currently remain in place and functioning: 2 cysts, 2 augmentations and 2 reconstructions following tumour surgery. The patients did not agree to the recommended bone implantation as they were satisfied with the quality of life ensured by the implant.Figure On the outer surface of the reference implant Fig. , the fibFigure Figure The implant retrieved from the human body was treated with an enzyme solution at 37 °C for 5 days in order to remove the remaining human soft tissues from the surface and thereby permit the study of the surface morphology. The inner and outer surfaces of the enzyme-treated implant proved to be very similar Fig. .Fig. 9ScSEM images of the cross section and the outer surface of the implant can be seen in Fig. In the low-magnification image of the cross section Fig. , the cavIn both the reference implant and the implant retrieved from the human body after 8 years, the measured thickness of the carbon fibres in the bunches was between 5 and 7 μm, in good agreement with the specification of the manufacturer of the carbon fabric. This suggests that the thickness of the carbon fibres did not change during the 8 years that the implant spent in the human body.In order to investigate the potential interactions between the implant and the human body, the average surface compositions of the reference implant and the implant retrieved from the human body were determined by the EDS method. The first two rows in Table Since the residual human soft tissues partially covered the surface of the implant retrieved from the human body, it was subjected to enzyme treatment before determination of the average surface compositions of the two sides. As the informal depth of EDS measurements on pure carbon with the parameters used is ~4.5 μm, only the average surface compositions of the 15–17-μm-thick surface layers on the two sides of the retrieved implant were determined. The measured compositions are to be found in rows 3 and 4 in Table Figure A number of questions were raised in the Introduction. We are now in a position to give answers to these questions as concerns the C/C implants employed in the present investigation.In connection with the first question, it may be stated that, in the cases (11 of the 16) when the Carbulat™ did not have to be removed before time, the result was very good from both a functional and an aesthetic aspect. When removal of the implant did become necessary before time, this was never due to any problem with the Carbulat™ as a material, but rather for various technical reasons: scar tissue with a poor blood supply, wound strain or wound opening. When the aesthetic results were compared with those achieved after the use of titanium reconstruction plates, the tissues proved to be better integrated around the Carbulat™ and the replacement with this material gave a better resemblance to the original shape and volume of the mandible.As concerns, the question of systemic toxicity, it must be reiterated that the findings of the investigations performed to satisfy the demands of the U.S. Food and Drug Administration were satisfactory and carbon is not known to have a toxic effect . In accoFrom the aspect of the immediate environment of the implants, we conducted macroscopic and microscopic examinations on two implants, which had functioned in human organisms for 8 months and 8 years, respectively Figs.  and 8. TOur examinations to date have revealed black carbon particles only in the immediate vicinity of the plate; we have never observed them farther away Fig. . We consTo determine how the long-term exposure to the harsh environment of the human organism changes the structure and morphology of the implants, we set out to study this question with SEM and EDS.The 5–7-μm-thick carbon fibres on the inner and outer surfaces of the reference implant Fig.  were fouBoth the inner and the outer surface of the implant retrieved from the human body were covered by a 15–17-μm-thick cracked layer, below which the carbon fibres were still visible. As our measurements indicated that the thickness of the carbon fibres (5–7 μm) did not change during the 8-year period spent in the human body, the material constituting this surface layer presumably does not originate from the carbon fibres. A possible source is the pyrolytic carbon introduced to the implant during the manufacturing.Cavities can be found between the perpendicularly oriented bunches in the implants. The cavities present in the implant retrieved from the human body were larger than those present in the reference implant. In the images of the retrieved implant Fig. , human sBesides the two main components (carbon and oxygen), small quantities of sodium, chlorine and zirconium were detected on the surface of the reference implant Table . The facThe effects of the human organism on the composition of the surface indicate that a slight extent of material transport occurs between the implant and the surrounding human tissues. It might prove beneficial to prevent this completely through the application of a stable and biocompatible surface thin layer.The clinical conclusion is that, if the soft part cover is appropriate, the carbon implants are cosmetically and functionally stable. The width of the carbon fibres (5–7 μm) building up the implants was not changed during the interaction with the human body. The structure and the morphology of the implants were not altered notably by the human body.Further research is required to establish the effect of the findings presented here on the life quality of patients with pyrolytic carbon-covered C/C composite implants. A possible solution to enhance the long-term biocompatibility properties of the implants is the application of a thin surface layer of a biocompatible and inert material.Written informed consent was obtained from the patient for the publication of this report and any accompanying images.


PVTt) primary gas flow standard at the National Institute of Standards and Technology has an expanded uncertainty (k = 2) of between 0.02 % and 0.05 %. The standard spans the flow range of 1 L/min to 2000 L/min using two collection tanks and two diverter valve systems. The standard measures flow by collecting gas in a tank of known volume during a measured time interval. We describe the significant and novel features of the standard and analyze its uncertainty. The gas collection tanks have a small diameter and are immersed in a uniform, stable, thermostatted water bath. The collected gas achieves thermal equilibrium rapidly and the uncertainty of the average gas temperature is only 7 mK (22 × 10−6T). A novel operating method leads to essentially zero mass change in and very low uncertainty contributions from the inventory volume. Gravimetric and volume expansion techniques were used to determine the tank and the inventory volumes. Gravimetric determinations of collection tank volume made with nitrogen and argon agree with a standard deviation of 16 × 10−6TV. The largest source of uncertainty in the flow measurement is drift of the pressure sensor over time, which contributes relative standard uncertainty of 60 × 10−6 to the determinations of the volumes of the collection tanks and to the flow measurements. Throughout the range 3 L/min to 110 L/min, flows were measured independently using the 34 L and the 677 L collection systems, and the two systems agreed within a relative difference of 150 × 10−6. Double diversions were used to evaluate the 677 L system over a range of 300 L/min to 1600 L/min, and the relative differences between single and double diversions were less than 75 × 10−6.A new pressure, volume, temperature, and, time ( PVTt systems have been used as primary gas flow standards by the National Institute of Standards and Technology (NIST) and other laboratories for more than 30 years  is chosen that falls late within the dead-end time. The match pressure value is found in both the start and stop data series and the time differences between the match pressure measurement and the start and stop trigger signals (Δti and Δtf) are determined from the data record. The appropriate time correction is added to the approximate collection time from the timers. Thus, by adjusting the collection time using the inventory data records, the initial and final inventory pressures and temperatures are nearly matched, leading to nearly equal initial and final inventory densities and inventory mass cancellation.As represented in First recall that the inventory sensors have non-zero time constants and therefore the measurements they provide are damped versions of the real conditions and further, the values they report at any given instant are subject to the recent history of the pressure or tempera ture value. Second, realize that perfect symmetry of conditions before and during diversion is unobtainable and that these imperfections and the significance of the sensor damping increase with the flow. For example, at high flows, the rate of change of pressure during the tank filling process is large and it becomes more difficult to make the pressure at which the stop diversion begins closely match the pressure at which the start diversion began . This “trigger pressure difference” will be considered again in Sec. 4. As another example, the bypass and tank valves may not close at the same speed.mI to a wide range of match pressure values.Analysis of the thermodynamic model of the inventory and its sensors shows that times later in the dead-end time give better mass cancellation under these circumstances since the sensor output enters a period with nearly constant slope that is equal to the real pressure slope. The experimental results given in mc indicates the mass of the high pressure cylinder and Vextra represents the extra volume temporarily connected to the tank for the purpose of introducing the gas from the cylinder to the tank . The extra volume is calculated from dimensional measurements or measured by liquid volume transfer methods.The volume of the 677 L tank was determined by a gas gravimetric method. In this method, the mass of an aluminum high pressure cylinder was measured before and after discharging its gas into the evacuated collection tank. The change in mass of the high pressure cylinder and the change in density of the gas in the collection tank were used to calculate the collection tank volume. Nominally,−6VT.In practice, a more complex formula than −6VT.The volume determination was conducted with both nitrogen and argon gas. In both cases high purity gas was used (99.999 %) and care was taken to evacuate and purge the system to minimize composition uncertainties. When nitrogen was used, the aluminum cylinder weighed approximately 4200 g when filled at 12.5, and approximately 3800 g after it was emptied to 55 kPa. When argon was used, the initial and final masses were 4440 g and 3820 g, respectively. The standard deviation of the six volume measurements  was 16 × 10S represents the scale reading, ref indicates the reference masses, ρair is the ambient air density where the measurements were conducted, and Vext is the external volume of the high pressure cylinder and its valve and fittings. The density of the ambient air was calculated from the barometric pressure, the temperature and humidity inside the wind screen, and an air density formula that includes humidity [The initial and final masses of the gas cylinder were measured using a substitution process with reference masses and a mass comparator enclosed in a wind screening box. The comparator has a full scale of 10 kg and resolution of 1 mg. The cylinder and a set of reference masses of nearly the same weight were alternated on the scale five times. The zero corrected scale readings were then calibrated to the reference masses and buoyancy corrected via the following formula:humidity .Tref) of 296.5 K with the following formula:α is the coefficient of thermal expansion for the aluminum tank. The terms containing α correct for changes in the cylinder volume due to differences between the water temperature, the air temperature, and the reference temperature. However, for this particular case, these thermal expansion issues could have been neglected since both the water temperature and air temperature never differed from Tref by more than 1.5 K. The thermal expansion corrections to the external volume were less than 0.5 cm3 or 100 × 10−6Vext and the external volume has a small sensitivity coefficient in the collection tank volume determination process.The external volume of the high pressure cylinder appears in 3 to 4709 cm3 between the 100 and 12.5 Mpa pressures. This change agreed well with predictions based on material properties, and the appropriate experimental values for external volume were used in the cylinder mass calculations [−6.The expansion of the external cylinder volume as a function of its internal pressure was not negligible. The Archimedes principle measurements showed a volume increase from 4697.5 cmulations , dependi−6 even though the two tanks were connected.The 34 L collection tank volume, the inventory volume for the large collection tank, and the small inventory volume were all determined with a volume expansion method. In this method, a known volume is pressurized, the unknown volume is evacuated, a valve is opened between the two volumes, and the density changes within the two volumes are used to calculate the unknown volume. Applying conservation of mass to the system of the two tanks yields:−6) and dimensional forms. The units of the dimensional values are given in the third column. The relative contribution of each sub-component to the combined uncertainty is listed in the fourth column. This contribution is the percentage of the squared individual component relative to the sum of the squares of all sub-components. The uncertainty from the inventory volume, the combined uncertainty, the expanded uncertainty, and the uncertainty contributions are given as a range covering the minimum to maximum flow. To calculate their relative uncertainty in The uncertainties contributing to the mass flow measurement have been quantified in −6−6At the highest flow, uncertainty contributions are principally divided between the tank volume, the final gas density, and the inventory uncertainty. The uncertainty falls to 200 × 10−6−6−6The performance of the flow standards and the validity of their uncertainty analyses  were checked by performing two types of experiments described in Secs. 4.1 and 4.2.To test both systems, we performed comparisons between the two flow standards over the range of flows where they could both be used: 3 L/min < −6PVTt systems should be less than the root sum square (RSS) of the uncertainties of the two standards, especially when one considers that the uncertainties due to pressure and temperature measurements are correlated between the two standards. For the lowest flows of the comparison range, the uncertainties originating from the inventory volume are quite small for both systems and the observed differences between them are dominated by tank volume uncertainties. From −6−6 (k = 1).How well should the two systems agree? The difference between the discharge coefficients measured by the two −6−6 (k = 1) contributed by the inventory volume at the highest flows in the 34 L system  was 20 times more important when using the smaller tank. m is the mass collected and t is the collection time using the 34 L tank. Making the assumption that there exists a constant error in the mass measurement, δm, for all flows (say due to a tank volume error) and a constant error in time measurement, δt, for all flows (say due to the time constant of the inventory pressure sensor), we can derive a simple linear model for the error in mass flow measurement, i.e.,−6Since the mass collected and the mass error are both essentially constant, this model suggests that a linear function of the inverse of the collection time should fit the tank comparison data. Such a linear function fits reasonably well, as shown in −6The comparison results could be the basis for corrections to the 34 L system. The intercept of During some of the comparison flows, we noticed that the pressure downstream of the critical flow venturi was significantly higher in the 34 L system than in the 677 L system (108 kPa vs 100 kPa) due to the smaller tube size and resultant higher pressure drop. For some of our venturis (with relatively short diffusers) this pressure difference caused slight changes in the upstream pressure (and the discharge coefficient), even at conditions well above the critical pressure ratio. Therefore, our assumption that for the same throat Reynolds number, the discharge coefficient of the venturi is independent of the downstream pressure may not be perfectly valid. We suspect that some of the differences observed between the tanks in −6 in discharge coefficient for each 1 kPa change in the trigger pressure difference. Since the largest trigger pressure difference is less than 3 kPa in the present system, this effect is expected to contribute only 30 × 10−6In one series of experiments, the trigger pressure difference was purposely varied over a range from −2 kPa to 27 kPa at a constant flow of 82 L/min in the 34 L system. The purpose of the test was to measure the dependence of the venturi discharge coefficient on the trigger pressure difference and hence assess its influence on the inventory volume uncertainties. The tests showed a relative change of 10 × 10two start and stop diversions. The intermediate dead-end times were set up so that the pressure transients in the inventory volume still permitted the mass cancellation procedure. Breaking the collection into two subintervals has the effect of doubling the uncertainty contribution from the inventory volume. The CFV discharge coefficients from the two protocols were compared to assess the magnitude of the uncertainties introduced by the inventory volume and the flow diversion process. Three flows between 300 L/min and 1600 L/min were tested and the differences in discharge coefficient were all less than 75 × 10−6To confirm that the uncertainty analysis for the inventory volume of the 677 L collection system was reasonable, we performed CFV calibrations at identical flows following two different protocols. In the first protocol, the inventory volume was dead-ended at the beginning and end of the collection interval, in the usual manner. In the second protocol, the collection interval was divided into two subintervals, i.e., each flow measurement had PVTt collection tanks (34 L and 677 L) has been presented. The system is designed to calibrate critical flow venturis for flows from 1 L/min to 2000 L/min.The design of a new gas flow standard composed of two The flow standard has several novel features. The collection tanks are immersed in a water bath that matches the nominal room temperature and is stable and uniform to 1 mK. The collection tanks are divided into sections of small enough diameter that the gas inside them achieves thermal equilibrium with the surrounding water bath in 20 min or less. This reduces the contribution of temperature to the flow measurement uncertainty to a low level.Uncertainties related to the inventory volume and the diversion of gas into the collection tank at the start and stop of a flow measurement have been studied in great detail. A thermodynamic model of the inventory volume during diversion has been utilized to understand the large pressure and temperature transients and the importance of sensor time constants on the flow measurement uncertainty. The flow standard is operated to achieve “mass cancellation” in the inventory volume, thereby taking advantage of correlated sensor uncertainties to minimize uncertainty contributions from the inventory volume. The uncertainty contributions of the inventory volume have been considered from both the time and mass perspectives.−6VT.The volumes of the collection tanks were measured by two methods, a gas gravimetric method and a volume expansion method. Six gravimetric determinations of the 677 L collection tank volume made with nitrogen and argon agree with a standard deviation of 16 × 10PVTt system and followed the propagation of uncertainties method suggested by international standards. The uncertainties of the sub-components have been examined at a fundamental level.A detailed uncertainty analysis for the gas flow standard has been presented in −6−6−6−6−6P) which is the major contributor to gas density and tank volume uncertainties. For air flow measurements using gas from the existing compressor and drier, mass flow uncertainties are about 500 × 10−6The uncertainty analysis shows that the 677 L system measures mass flow with an uncertainty between 200 × 10−6−6Comparisons between the 34 L and 677 L standards from 3 L/min to 100 L/min show agreement within 150 × 10PVTt flow standards. Pressure uncertainties are the most significant contributors, through the tank volume determinations as well as through the final gas density measurement. Therefore, tank pressure sensors with better calibration stability over time will be sought. Faster inventory pressure sensors would improve the mass cancellation procedure and reduce the inventory uncertainties.There are opportunities for improvement in the uncertainty of the new


Caenorhabditis elegans as an object for transmission electron microscopy makes its introduction into autophagy research a challenging task. The basis of the protocol to prepare C. elegans samples for autophagy studies was worked out around the turn of the millennium and has been used since then in my laboratory with some modifications. The method described here enables the user to prepare samples for systematic morphologic as well as morphometric investigations to characterize autophagy with a high but still realistic investment of effort.Traditional ultrastructural characterization of autophagic processes remains an important approach to be used in parallel with molecular genetics, light microscopy, and other methods. The special nature of  The recent exponential growth of interest in autophagy is taking place in an era of unprecedented progress in methodology of cellular and molecular biology and genetics. While it is the new approaches which are the driving force behind the rapid development of our knowledge, many traditional methods still remain important. Due to the small size of subcellular structures participating in the autophagic process, transmission electron microscopy (TEM) maintains a prominent position in the methodological arsenal for studying autophagy. Innovations involving TEM  have also been and are being developed. They may also be necessary for investigating certain aspects of autophagy; however, their exceedingly high investment needs  make their routine use unrealistic. The traditional TEM approach, however, is at the level of effort which makes it possible to apply it as a tool for regularly and systematically collecting qualitative and quantitative data on subcellular processes (including autophagy) in various cell types.The sample preparation procedure is one of the key factors for successfully studying cellular structures by TEM. The final result may depend on nuances of the applied technique, which are not written down in sufficient detail in the usual research articles. In addition there are special requirements of sample handling which, to a large extent, are dependent on the properties of the particular type of object we want to study.C. elegans is rather special for studies by TEM in itself . The other feature is that their cuticule, which covers the whole body, is practically impenetrable to the fixative, which has to diffuse into the cells to carry out its function of properly preserving intracellular structures. The easiest way to overcome the problem of penetration is to cut the worm into pieces in a drop of fixative under a dissection microscope. This action requires higher than average manual skills and makes it necessary, and at the same time possible, to treat the worms individually. Although cumbersome, the need for individual treatment during cutting offers the advantage of allowing observations in single worms which, thereby, can serve as the natural units for both qualitative and quantitative results, and makes it possible to reveal variations among the individuals as well as to carry out proper statistics for morphometry.There are two main features of C. elegans consists of a relatively small number of cells. There are 959 somatic nuclei in an adult hermaphrodite, and consequently considerably less in its various tissues. This allows one to observe characteristic features in the whole worm at the level of individual cells by light microscopy due to the transparency of the body wall. The situation is basically different in TEM studies in which sections must be prepared to reveal the structures inside the cells. Cross sections of the worm’s body are circles which offer only very small areas from each tissue. In addition, as the structure of the body is continuously changing from the tip of the head to the end of the tail, each cross section represents only a certain narrow region. To cover the whole body by cross sectioning would be exceedingly laborious and time consuming for a routine method in autophagy studies. This problem can be overcome by longitudinal sectioning along the antero-posterior axis, which requires proper and exact orientation of the samples. Even in this case, however, we apply serial sectioning. In our method, a set of sections covering five grids satisfy the needs of both qualitative and quantitative observations, giving a good overview of autophagic events at all developmental stages, and in all major cell types of the body   or cut  pieces of razor blades because they are thinner and apply less mechanical pressure on the body during cutting : it is possible to use a wide variety of recipes, presently we apply 3.2% formaldehyde, 0.2% glutaraldehyde in 0.15 mol/L sodium cacodylate buffer, pH 7.2Caution: even at the applied relatively low concentration, aldehydes are irritants and cacodylate is toxic, so avoid contact with skin, especially take care of eyes, and work in a well ventilated room.Washing buffer (WB): 0.125 mol/L sodium cacodylate buffer, pH 7.20.1% ruthenium red solution in DWDissecting stereo microscopeInverted microscope with digital cameraTP with wetted insert of artificial sponge or stack of filter paperWetting solution: 1.5% glutaraldehyde in DW to prevent fungal and bacterial growthAdjustable pipettesObliquely cut fine plastic pipette tip (PT) with good transparencyHair device: a piece of hair inserted and glued into the tip of a Pasteur-pipette or fine plastic PT; Pipette out 100 µL fixative into a PD. On an appropriately hydrophobic surface this volume can spread out as a 10 mm diameter convex drop.Put a single worm into the drop of FB. In fixatives with high formaldehyde content the worms stop wriggling within 10–20 s, while in solutions with glutaraldehyde only, they may keep moving for many minutes.If necessary a photograph of the intact worm in the fixative can be easily made at this stage using an inverted microscope. This may facilitate later orientation inside the TEM sections and the identification of corresponding structures at light microscopy and TEM levels.Cut the worm into maximum 3–4 pieces.Collect the pieces in one group in the center of the drop with the help of the hair device.Using the obliquely cut fine plastic PT, pipette up all pieces with one draw if you can, and release them into the appropriate well of the TP. Check if all pieces are transferred. Repeat the process with pieces left behind.If a piece (or more) is missing it is most likely attached to the wall of the PT. If the PT is transparent enough you can check that in the stereo microscope. This is usually not necessary though, as you can go back to the big drop of FB where you made the cutting, and release the attached piece by vigorously pipetting up and down. In rare cases when a piece attaches to the inside of the PT so tightly that it cannot be detached by pipetting up and down, you can release it under the microscope with the help of the hair device.Always keep track of all the pieces to avoid mixing of samples. If a piece happens to be lost in spite of all efforts, change both the PD with the FB and the used PT to new ones.Fill the well of the TP with fixative to make the surface of the solution slightly convex to act as a lens that makes it easier to recognize the samples.At this stage, we can collect the samples into the center of the well and make photographs again of them. This may be very useful to help in their identification, as the danger of losing a piece is always present in several of the later steps of sample preparation, especially during washing and embedding into agar.Add a few drops (1–2 µL) of 0.1% ruthenium red solution to the fixative until the buffer becomes deep red with the samples remaining clearly visible . In our experience ruthenium red has the advantage of making the sample pieces better recognizable during the whole preparation procedure.Let the samples stand in the fixative for a few (at least two) hours, or overnight in a refrigerator.Wash the samples with WB. This is done by replacing FB with WB, pipetting down as much of the FB as possible without removing any of the sample pieces. To make washing more efficient, collect samples with the hair device to one side of the bottom of the well, and use a plastic PT with a narrow capillary ending. In one washing round make three exchanges of WB and, altogether, carry out at least three rounds with at least some hours in between. In cacodylate-based WB, fixed samples can be left in the refrigerator for weeks without apparent change. Cacodylate prevents microbes growing in the WB, which may easily happen for example in phosphate buffers during longer storage.The next steps of sample preparation require serial changing of liquids which in TPs would be exceedingly time consuming, very inconvenient, or from a certain point, even impossible. This is partly because the worm pieces are very small, and also because several of the applied materials are either toxic, very volatile, or even aggressive solvents, which polystyrene cannot withstand. To overcome these difficulties, fixed pieces must be embedded into an appropriate material which keeps the cut parts together and, at the same time, allows the treating agents to properly carry out their action. The usual material for this embedding is either agar, or agarose. They are applied at a concentration which forms a strong enough gel at room temperature for keeping together the pieces against the mechanical forces of sample handling.We have routinely used 1.5% bacteriological agar liquefied by heating and kept in the water bath of a heating thermostat at 55 °C.In the stage of agar embedding we included an extra step to achieve the proper orientation of the samples for later longitudinal sectioning while using the most popular flat molds for resin embedding General purpose microbiological agar , its bottom is hydrophilic and the agar can be spread more thinly and easily on it 55 °C water bathThin strips of any type of common, general purpose cellulose filter paperDissecting stereo microscopeHypodermic needle of approximately 12–14 gaugeSmall (~10 mL) flat bottomed, clear, glass injection vials  with wide opening  which can be tightly stopperedOrganic solvent (ethanol and propylene oxide)-resistant test tube caps Graphite pencilAdhesive white paper Adjustable pipettesHair deviceRazor bladePrepare appropriate amount of 1.5% agar solution in DW and keep it in the 55 °C water bath.Pipette out 1.5 mL warm agar and spread evenly on the bottom of a 5 cm PD by swirling movement until it becomes a firm thin layer.Divide the PD into four quarters by drawing two perpendicular lines on the outside of the bottom, to make areas for four samples. Write the sample number in each quadrant, taking care to leave the central area open.Under the dissecting microscope pipette out the sample pieces from the TP into the appropriate quadrant, close to the center, with 1 µL WB. Take care not to lose any piece.Group the pieces to the edge of the drop by the hair.Remove most of the liquid with a thin strip of filter paper, and group the pieces with the hair device close together so that they touch one another. Remove the rest of the liquid by filter paper again.From a PT, which is trimmed to widen the orifice, let a drop of approximately 15 µL agar fall on the group of samples. Take care that the center of the drop hits the center of the sample group, otherwise the pieces can move away from one another.It is very useful to make a photograph of each sample at this stage.Cut out the sample in a small cube of agar with a razor blade [see also  and place it on a free agar area in the same quadrant.Turn the cube by 90° to make the plane of samples perpendicular to the fresh agar surface.Cover the turned agar cube with the samples inside it by adding a new drop of agar.Cut out a block of approximately 2 × 4 mm flat prism with the sample close to one of the edges [see also  into the numbered 8 mL vials with WB, stopper and put them in a refrigerator.As the worm pieces are inside the agar prisms, we can now handle our samples similarly to ordinary tissue blocks. The procedure is carried out by sequential changing of solutions over the agar prisms inside the glass vials. Various solutions for postfixation (staining) dehydration and infiltration are sucked off and the new ones are added. During the process, the small glass vials are put into a slow movement (2 r/min) rotation drum. The whole procedure up to the polymerization must be performed in a ventilated cabinet with good air suction. It is also advisable to use gloves during these processes.To make thin enough sections from the samples is a prerequisite for utilizing the high resolution of the electron microscope. Sections of approximately 60–90 nm thickness must be prepared to achieve our goal. This requirement is fulfilled by embedding into plastic polymers (embedding resins) which are highly hydrophobic. Therefore, the samples must be dehydrated before getting into the embedding resin. Dehydration is carried out as a stepwise process where a series of ethanol solutions with increasing concentration up to absolute alcohol is changed sequentially over the samples. Finally, propylene-oxide is applied to chemically remove even traces of water. This is followed by the infiltration with the resin. The infiltrated agar blocks are then transferred into the cavities of flat silicone embedding molds and put into a hot thermostat to polymerize.Ventilated cabined with efficient air suction4 solution in cacodylate buffer at pH 7.2 0.5% OsO1% uranyl acetate in DWSlow speed rotation drum for ~30 samples adjusted to 45° angleNarrow stemmed, bulbed, plastic transfer pipette which is organic solvent-resistantEthanol, propylene oxideTAAB embedding resin kit with DMP-30 (TAAB Embedding resin kit TK3 T004)ToothpicksMultispecimen, flat, silicone embedding molds (TAAB Embedding mould type B E071)60 °C degree thermostat4 solution for postfixation (staining) of about 1 h. The vials must be tightly stoppered because the osmic acid vapors are very volatile and toxic.Remove the WB using the transfer pipette and replace it with 1 mL 0.5% OsO4 solution and wash twice with DW .Remove the OsOReplace DW with 1% uranyl acetate in DW (~1 mL) for the next postfixation (staining) step, soak for 30 min, and also use the stoppers here.Apply a graded series of 50%, 70%, 96% and absolute ethanol (~2 mL in each case) for initial dehydration, leaving the samples in each solution for 20 min, changing the absolute ethanol twice. Stoppering is not necessary until you begin to use the absolute ethanol.Replace absolute ethanol with propylene oxide and leave it on for 20–30 min.Prepare the embedding resin before the end of dehydration, at an appropriate time. Many types of embedding resins are in use and can work well. We presently use the “TAAB embedding resin kit with DMP-30” with the following proportion of the components for 10 mL resin: TAAB resin 5 mL, DDSA 3.8 mL, and MNA 1.2 mL.Mix the components thoroughly to have a homogenous solution, add 0.2 mL DMP and mix well again.Dilute the above resin with propylene oxide by adding one part propylene oxide to three parts of resin.Replace the propylene oxide with the diluted resin of ~1.5 mL for initial infiltration, and leave the samples in the rotating drum for 4–5 h. Use stoppers!Prepare resin with the same proportion of components as above before the end of the initial infiltration, at an appropriate time, and replace the propylene oxide-resin solution with ~1.5 mL of this freshly prepared pure resin.Leave the vials containing the samples in the resin in the rolling rotation drum overnight. We cover the whole bunch of vials with a sheet of aluminum foil and do not use stoppers to facilitate the escape of traces of propylene oxide.The next morning, transfer the infiltrated agar blocks into the cavities of the embedding mold.Prior to placing the blocks into the cavity of the mold, take some of the resin from the vial with a transfer pipette and fill the cavity to more than half with it.Fish out the agar block with a toothpick and place it into the cavity containing the resin that has been put in it beforehand.Fill the cavity by adding more resin to make the top level with the plane of the mold, or slightly convex.Check the position of the blocks under a dissecting microscope when all the samples are in the cavities, and arrange the plane of the sample pieces parallel with the upper side of the cavity . This is important to ensure the precision of the later longitudinal sectioning.Place the molds into a 60 °C thermostat for polymerization for 2–3 days.C. elegans and are almost exactly the same as those of ordinary tissue pieces. The plastic blocks with the embedded samples must be trimmed to allow them being accessed for later sectioning. We do this by hand with a razor blade under a dissecting microscope (Sigmond et al. The remaining steps of sample handling are not special for The quality of embedding can be checked partly, when manual trimming is being done, and partly when the first sections have been made in the ultramicrotome. Sometimes samples may appear softer than optimal, then putting them back at this stage into the 60 °C thermostat for a couple of days can improve their quality.Sections are collected to cover five grids and stained in alkaline lead citrate solution prepared according to Reynolds Reynolds . Sometim


To increase the precision of estimated item parameters of item response theory models for patient-reported outcomes, general population samples are often enriched with samples of clinical respondents. Calibration studies provide little information on how this sampling scheme is incorporated into model estimation. In a small simulation study the impact of ignoring the oversampling of clinical respondents on item and person parameters is illustrated.Simulations were performed using two scenarios. Under the first it was assumed that regular and clinical respondents form two distinct distributions; under the second it was assumed that they form a single distribution. A synthetic item bank with quasi-trait characteristics was created, and item scores were generated from this bank for samples with varying percentages of clinical respondents. Proper  and improper (ignoring oversampling) approaches for dealing with the clinical sample were contrasted using correlations and differences between true and estimated parameters.Under the first scenario, ignoring the sampling scheme resulted in overestimation of both item and person parameters with bias decreasing with higher percentages of clinical respondents. Under the second, location and person parameters were underestimated with bias increasing in size with increasing percentage of clinical respondents. Under both scenarios, the standard error of the latent trait estimate was generally underestimated.Ignoring the addition of extra clinical respondents leads to bias in item and person parameters, which may lead to biased norms and unreliable CAT scores. An appeal is made for researchers to provide more information on how clinical samples are incorporated in model estimation. In the last decade computerized adaptive testing (CAT) has become a popular method for efficiently assessing patient-reported outcomes (PROs). For example, in the USA, the patient-reported outcomes measurement information system (PROMIS), a group of scientist from several academic institutions and the National Institutes of Health , has devθ. The items usually have two types of parameters: parameter a represents the extent to which the item is able to discriminate between different levels of the latent trait; parameter b is a location parameter which is expressed on the same scale as the latent trait and indicates the position on which a higher response category is preferred over a lower response category. The latent trait scale is arbitrary by definition ),θs were sampled. In the simulation, the general population had a normal distribution with a mean of −2 and SD of 1; the clinical population had a normal distribution with a mean of 0 and SD of 1. The percentage of clinical respondents was controlled by varying the number of draws from both distributions; for example, in the 50 % condition, 750 θs were sampled from each population.Under the first scenario, the general and clinical populations have distinct but overlapping distributions of the latent trait . For soθs were sampled from each region. It should be noted that prevalence should be distinguished from the rate of clinical respondents in the calibration sample, as the former rate is associated with a population and the latter with a sample. One of the goals of the current simulation is to show how estimates may be biased if the two rates are unequal due to the sampling scheme.Under the second scenario, the latent trait scores of clinical and general populations can be described by a single distribution; respondents scoring above a critical value suffer from pathology and thus belong to the clinical group . The crFor each scenario, the proper way of dealing with the clinical sample was compared with a default approach which ignored the sampling scheme. Under this approach, GRM was run for the entire matrix of simulated item response data in each calibration set, i.e., for all 1500 simulees and 40 items. Location and discrimination parameter estimates were obtained using marginal maximum likelihood , which aθ, one for the general population and one for the clinical population. To allow for a metric comparable to that of the true item parameters, the mean of the clinical population was fixed at zero, and the general population value was freely estimated; SDs of the latent trait in both populations were assumed to be equal to 1.Under the first scenario, the proper way of dealing with the clinical sample would be to fit a multi-group model . It is Under each of the two scenarios, two types of calibrations  were performed for each percentage of clinical respondents , resulting in a total of 12 sets of item parameter estimates. Models were estimated using the mirt library , 35 in RTwo outcomes were studied: recovery of item parameters and recovery of person parameters. To study the recovery of item parameters, the estimates resulting from each calibration were contrasted with the true  parameters. To that end, two measures were used: the correlation between the true parameter values and their estimates, and the mean difference between the true parameter values and their estimates.θ-values from −4 to 4 with an increment of 0.50, and item responses were created as described above. Next, these responses were used to estimate θs under each of the twelve calibrations. Estimation was performed using maximum a posteriori . Five hundred subjects were generated at each of 17 evenly spaced true ri , which b1 and lowest for the b4 parameters. Under the correct approach (the two-group model), the estimates were close to the true parameters . By contrast, under the incorrect approach (the one-group model), the parameter estimates were clearly biased . All a parameters were overestimated (the mean difference between true and estimated as was negative), whereas the pattern for the location parameters varied for the different levels of percentage of clinical respondents. For example, for b4 the difference was negative in the 25 % clinical respondents condition and positive in the other two conditions. Clearly, under the improper calibration approach the scaling of the latent trait was different from the true scaling. The incorrect assumption of a single distribution yields a scale with an origin which lies in between the means of the two true distributions and a unit which is about equal to the overall standard deviation of the combined populations.1Table θ as a function of true θ. Under the one-group  model approach the latent trait was overestimated substantially for most parts of the scale, except for the upper extreme region, where some underestimation was found; the size of the bias decreased with increasing levels of percentage of clinical respondents. In addition, to provide some meaning to the size of bias, let us inspect the estimates at a true θ of 2, as it is the analog of a T-score of 70 (corresponding to a percentile rank of 98), a common critical value in clinical assessment [θ was obviously too high , and severity was therefore overestimated, whereas in the 75 % condition bias was almost absent.The upper panel of Table sessment . In the θs ranging from −1.5 to 4.0, but for θs below −1.5 estimates were too high, and bias increased with decreasing θ. The bias on the lower side of the scale resulted from the item bank having little information for this region. As a result much weight was given to the prior distribution employed by MAP, which pulled the estimates toward zero (a phenomenon commonly known as ‘shrinkage’).Under the two-group  model, the estimated values were unbiased for θs in Table Examining the standard errors of the estimated b4 parameter. Under the proper approach  the item parameters were unbiased, having mean differences of about zero. By contrast, under the improper approach biased estimates were found. The a parameter was clearly overestimated in the 25 and 50 % condition, whereas it was slightly underestimated in the 75 % condition; the location parameters were underestimated in all three conditions, with bias increasing with increasing levels of percentage of clinical respondents. The incorrect assumption of random sampling yields a scale with an origin which lies at the right-hand side of the true mean and a unit which is about equal to the standard deviation of the unweighted true distribution.Table θ as a function of true θ.The lower panel of Table θ < −1.5). Under the model without weights  the latent trait was mostly underestimated, except for the lower extreme region, where it was overestimated; bias increased with higher rates of clinical respondents, with substantial values in the 50 and 75 % conditions. In addition, to give more meaning to the size of bias, let us again inspect the estimates at a true θ of 2 (comparable to a T-score of 70). In all three conditions the mean estimate of θ was obviously too low , and severity was therefore underestimated.Under the model with weights , patterns were highly similar to the two-group model of the first scenario in that the latent trait was unbiased except for the lower left part , rate of clinical respondents in the calibration sample, and the true value of T-scores, may be too high or too low, which may lead to over- or under-diagnosing of complaints. In addition, it also showed that scores may not only be biased but may also be less reliable if adaptive testing is used. As the standard errors calculated under the improper calibrations were generally too low, and the size of the standard error is commonly employed as stopping criterion in CAT, stopping may occur too early resulting in scores that are less reliable than required.This study showed that ignoring the sampling scheme may lead to structural over- or underestimation of item parameters and therefore to biased latent trait estimates. As a consequence, the norm-scores used in clinical practice, such as percentile ranks and a parameter  in the latent trait estimate. In addition, overestimation of discrimination parameters was associated with underestimation of the standard error of the latent trait. As the amount of test information is known to increase with higher values of the er , it is θ < −1.5. This is a result of the combination of an item bank measuring a quasi-trait and the Bayesian nature of MAP estimation. By assuming a prior distribution, the estimates were pulled toward the center of the population distribution [Even when the sampling scheme was properly dealt with, the latent trait was generally overestimated for ribution , zero inribution . It may ) .In the current study, data were generated and evaluated under two popular perspectives on the nature of the latent trait. Although both views allowed for generating item responses which resembled those typically found for quasi-traits, this pair of perspectives is not comprehensive. For example, under both views normal distributions were assumed, whereas recent research has suggested that some latent traits underlying PROs may be skewed , 42 or uThis paper shows the importance of properly dealing with extra clinical samples and it is recommended that future calibration studies provide more information on the approach taken for dealing with the issue so that its appropriateness may be evaluated.


Within cells, stimuli are transduced into cell responses by complex networks of biochemical reactions. In many cell decision processes the underlying networks behave as bistable switches, converting graded stimuli or inputs into all or none cell responses. Observing how systems respond to different perturbations, insight can be gained into the underlying molecular mechanisms by developing mathematical models. Emergent properties of systems, like bistability, can be exploited to this purpose. One of the main challenges in modeling intracellular processes, from signaling pathways to gene regulatory networks, is to deal with high structural and parametric uncertainty, due to the complexity of the systems and the difficulty to obtain experimental measurements. Formal methods that exploit structural properties of networks for parameter estimation can help to overcome these problems.We here propose a novel method to infer the kinetic parameters of bistable biochemical network models. Bistable systems typically show hysteretic dose response curves, in which the so called bifurcation points can be located experimentally. We exploit the fact that, at the bifurcation points, a condition for multistationarity derived in the context of the Chemical Reaction Network Theory must be fulfilled. Chemical Reaction Network Theory has attracted attention from the (systems) biology community since it connects the structure of biochemical reaction networks to qualitative properties of the corresponding model of ordinary differential equations. The inverse bifurcation method developed here allows determining the parameters that produce the expected behavior of the dose response curves and, in particular, the observed location of the bifurcation points given by experimental data.Our inverse bifurcation method exploits inherent structural properties of bistable switches in order to estimate kinetic parameters of bistable biochemical networks, opening a promising route for developments in Chemical Reaction Network Theory towards kinetic model identification.The online version of this article (doi:10.1186/s12918-014-0114-2) contains supplementary material, which is available to authorized users. In the context of systems biology, modeling is used to drive the acquisition of knowledge about the molecular mechanisms governing intracellular processes by evaluating the system’s behavior. In this way, measuring how the levels of some key species in the network evolve, for example, upon environmental perturbations, may help to retrieve important features concerning connectivities and rate constants of the underlying mechanism . One key challenge in systems biology is the identification of kinetic models from limited quantitative data ,2, hencetake decisions, by transforming, for example, graded stimuli into all or none responses. Bistable switches are ubiquitous in cell signaling networks, from pheromone sensing [Biochemical switches are biochemical reaction networks with the capacity for two different stable steady states. Depending on the initial and environmental conditions the system will evolve towards one steady state or the other. This property enables the cell to  sensing  to cell  sensing . The pre sensing ,9, which sensing .How can bistability bring insight on the parameters of a model?forward problem of determining the mapping from the parameter space to the space of the model behavior and, in particular, to the capacity of the model for bistability, can be approached using standard bifurcation analysis [The analysis  when theanalysis  a conditThe inverse problem of finding parameter regions compatible with bistable behaviour can be tackled by checking the multistationarity condition derived in  through a to infer the kinetic parameters, exploiting the condition derived in [Here, our goal is to exploit quantitative information from experimental data in order to infer the actual values of the model parameters, and what we develop is a method for parameter estimation from experimental data. Typical evidences for bistability obtained from experiments are, for example, hysteretic dose response curves. Dose response curves indicate how the steady state of a system evolves upon increase or decrease of a given stimulus. In a hysteretic dose response curve a continuous change in the stimulus (dose or input), produces a discontinuous jump in the measured steady state concentration or output beyond the so called bifurcation point. In this work we address the inverse problem of determining the parameters that produce certain desired properties of the bifurcation diagram and, consequently, in the observed experimental dose response curves. In particular, the method uses the location of the bifurcation points given by experimental datarived in . In contrived in ,14 and cThe basic ingredients of the inverse bifurcation method are described in the Methods section, where a brief introduction of some essential CRNT concepts is given together with the condition for multiple steady states derived in , here exm the number of species and by r the number of reactions in a reaction network, the balance equations describing the dynamics of the concentrations of the species involved are a set of ODEs usually written as b. Within the context of CRNT alternative ways to encode the balance equations are used such that the connection between structure and dynamics can be more easily exploited [We consider biochemical reaction networks with mass action kinetics where the dynamics are modeled by systems of ordinary differential equations (ODEs). The structure of mass action kinetic models is given by the connectivity and the stoichiometry of the network and the parameters are the kinetic rate constants. Let us denote by xploited . Resultsxploited , which iS to its active form P is mediated by another protein or enzyme E, whereas S inhibits its own activation. The complexes of the network are the linear combinations of species at both sides of the reaction arrows and the number of complexes is denoted by n. In the graph of complexes of a reaction network the nodes are the complexes linkage classes and the number of linkage classes is denoted by ℓ. Two nodes strongly linked if there is a directed path from terminal strong linkage class is a maximal set of nodes within a linkage class such that there is no edge pointing to any other set of nodes strongly linked. The number of terminal linkage classes is denoted by t. Networks with t>ℓ are considered in general to be unsuited for the description of real chemical systems [t=ℓ .For illustrative purposes we consider the mechanism in Figure  systems . In whatFor a reaction network being bistable there is necessarily an exchange of matter and/or energy through the boundary of the system. According to the second law of thermodynamics isolated systems evolve towards the thermodynamic equilibrium. Isolated reaction networks endowed with mass action kinetics achieve the thermodynamic equilibrium which has been demonstrated to be globally asymptotically stable ,16 preclS→2S, or explicitly in the form of pseudo reactions of the species that are entering or going out of the control volume with a zero complex representing the environment. In the context of signaling, we use the same formalism of pseudo reactions with the environment to account for the formation or degradation of a species S (∅→S or S→∅ respectively).Exchange of matter can appear implicitly, as it happens in reactions of the form S and P, and the constitutive formation of the inactive form S. The corresponding graph of complexes is depicted in Figure i from 1 to n.In our example we consider the degradation of the species yij is the molecularity of the species i in the complex j=1,…,n has an associated mass action monomial: Once species and complexes are labeled (note that the ordering of both the species and the complexes is arbitrary) we build the molecularity matrix kjk. The corresponding reaction rate is vjk(c)=kjkψj(c). Within this framework the balance equations read: and every arrow in the graph, going from complex where the matrix onstants .stoichiometric compatibility class, which is next defined after introducing the stoichiometric subspace. The stoichiometric subspace is the subspace spanned by the columns of the stoichiometric matrix N. Considering the formulation in Eq. TB=0. Let us take a reference concentration vector c0 and define As a consequence of the mass conservation laws the trajectories of Eq.  in the cn in Eq. , it can reaction polyhedron consists of all c≥0 such that W=0. Closed reaction networks are conservative systems [v (with m elements) such that vS=0. On the contrary, open systems are not conservative. The existence of conservation laws implies that the ODE system . The derivation of the set of ODEs . For networks with t=ℓ the condition in [t=ℓ networks. The reader can find the detailed derivation and proof in [t=ℓ networks follows.A condition for multiple steady states in biochemical reaction networks is derived and demonstrated in  for weakition in  is stillproof in , from whc that satisfies YAψ(c)=0. Equivalently, one can say that a vector z=Aψ(c) belongs to the deficiency subspace: The system in Eq.  is at eqdeficiency of the network, δ=dim(Dδ). For networks with t=ℓ the deficiency is given by the formula δ=n−ℓ−s, and a basis ω1,…,ωδ for the deficiency subspace Dδ can be computed from graph matrices taking into account that: The dimension of this subspace is the Λ is a n×ℓ matrix in which entry Λij= corresponds to the node Λij=1 if Λij=0 otherwise.where z defined above is a linear combination of the vectors of the basis of Dδ, we have: Since the vector ψ(c)>0, we get: On the other hand, from Eq.  providedn−ℓ linearly independent equations can be obtained in terms of the state vector From Eqs.  and 8),,8), a sek. Eq. =0, where W has been introduced in Eq.  and y is the magnitude of the associated system’s response.e curves , are x-yobservable) is plotted against some parameter (input). The parameter can be a kinetic rate or a conservation law constant, i. e. the total amount of an enzyme or conserved moiety, bi in Eq.  enclosing the region where bistable behavior is observed.For illustrative purposes we focus first on structures where cl=c and cu=c where the tangent bifurcation condition is fulfilled. Therefore, we have: c>0, c>0 belong to the equilibrium manifold, i.e.: The two points defining the lower and upper bound of the region of bistability in the dose response curve indicate two different stoichiometric compatibility classes tangent to the equilibrium manifold. They correspond, in fact, to two different points in the state space n by Eq.  and c: In order to efficiently search for a point in the space of the kinetic parameters satisfying all the conditions above we can solve an optimization problem. First, we define the objective function to be minimized  or combinations of concentrations (ql and qu) at the bifurcation points are obtained from dose response curves. This optimization problem is non convex and multimodal and consequently a global optimization algorithm [where lgorithm  is used k which is compatible with the observed dose response behavior. It is important to note here that, although we obtain a single point k in the parameter space as the output of the global optimization algorithm, the solution of the optimization problem  and, on the other hand, the degrees of freedom which are needed to define the equilibrium manifold at the two bifurcation points. For networks with λ=δ we take the deficiency parameter vectors λ≠δ a total of 2λ variables are taken from the vectors of deficiency parameters We define the A first set of  constraints is given by: with The mass conservation laws and the observed concentrations constitute the third set of  constraints: bl and bu, as well as the corresponding concentrations (cl and cu) or combinations of concentrations (ql and qu) at the bifurcation points are obtained from dose response curves. Finally, a set of  constraints is given by where the values of pL and pU are lower and upper bounds for the decision vector variables.where ki, two extra equality constraints need to be satisfied, indicating the values of the manipulable parameter at the lower and upper bifurcation points : In order to find the regions in the parameter space where the constraints -17 are set al. . This alsubject to the constraints already defined by -17. Thisa priori starting from a given set of observables, by evaluating the viable regions obtained.The exploration algorithm provides as an output a large set of uniformly distributed viable points that allow to characterize the viable regions in the parameter space, i.e. those regions in the space of parameters that fulfill the constraints imposed . The larS, ESS and P. The corresponding values are included in Table ρ1,…,ρ7 (including rate constants and/or combinations of rate constants and deficiency parameters) is obtained. These identifiable parameters are shown in Table We use this approach in order to find a set of identifiable parameters in the protein activation network model. We take as observables the total enzyme concentrations at the lower and upper bifurcation points, mentclass2pt{minimThe bifurcation diagrams and tangent bifurcation points computed for the viable parameter regions are depicted in Figure k from the datae.In a real experimental scenario we expect to get a number of replicate measurements for each data point which are subject to experimental error, and we need to estimate the unobserved kinetic parameters k, and εbl and εbu represent vectors of residuals. Similarly, the expressions for the concentration observables read: Let αl and αu (in this expression α1=αl and α2=αu): We can express the sum of squares of the residuals between the observed responses in the data set and the model prediction as a function of the kinetic parameters and deficiency parameters J1 defined in Eq.  are measured at these values of the decay constant. The in silico experimental dose response data  are shown in the figures contained in the Additional file From the dose response measurements, two tangent points are localized, at low and high decay constant values entclass1pt{minimaG1T=G2T=0.16605, computed from the gene copy numbers and the cell volume.The gene regulation network has two mass conservation laws  the ‘tangency’ condition from the reaction graph, used here instead of a generic bifurcation condition. On the other hand, the method uses the insight that tangent bifurcation points are precisely the loci where the equilibrium manifold and the reaction simplex become tangent. The tangency condition is an algebraic expression dependent on the parameters that can be systematically computed from the graph matrices. In this way our method avoids simulating the system at every iteration of the optimization algorithm reducing dramatically the computational effort in comparison to direct fitting .a priori and select a set of parameters which are identifiable, we propose a constraint satisfaction problem. This problem is numerically solved by an algorithm [a priori a set of identifiable parameters is subject of future work.As a first approach to assess the identifiability lgorithm  that allIn presence of experimental uncertainty, Monte Carlo simulation and global optimization are combined to generate the parameter distributions compatible with the experimentally observed behavior.In this work we show how a set of parameters can be identified using only information about the location of the bifurcation points in the dose response curves. The inverse bifurcation method could also be applied in combination with standard approaches , to significantly improve identifiability and facilitate the parameter estimation task.The method has been successfully applied to a number of examples of interest in biology including a protein activation network and a gene regulatory toggle switch, illustrating the potential of the Chemical Reaction Network Theory for kinetic model identification.a In this paper we use experimental data generated in silico.b The time derivative x depending on time t is denoted by n dimensional real vectors is denoted by m×n real matrices by n dimensional real vectors with all strictly positive entries is denoted by n dimensional real vectors with all nonnegative entries by c In what follows we denote the null space (or kernel) of a matrix X by Ker(X).dDxF denotes the Jacobian of F with respect to x.e We here assume for simplicity that the set of parameters k is identifiable. In case a different set of parameters ρ is found to be identifiable a priori the decision vector k is replaced by ρ.


A free-floating wave energy converter (WEC) concept whose power take-off (PTO) system reacts against water inertia is investigated herein. The main focus is the impact of inclining the PTO direction on the system performance. The study is based on a numerical model whose formulation is first derived in detail. Hydrodynamics coefficients are obtained using the linear boundary element method package WAMIT. Verification of the model is provided prior to its use for a PTO parametric study and a multi-objective optimization based on a multi-linear regression method. It is found that inclining the direction of the PTO at around 50° to the vertical is highly beneficial for the WEC performance in that it provides a high capture width ratio over a broad region of the wave period range. Energy is thus extracted from the relative motion between the piston and the tube. This concept was originally pioneered by the Swedish company Inter Project Services AB whose device (the IPS buoy) was fitted with a vertical PTO tube as in figure 1a , the optimization will only be conducted with values of m2≤m1. Extending the range of m2 helps ensuring the validity of the models for values of m2 close to m1. The upper m2 value retained for the optimization (m2=m1) would correspond to a tube whose internal volume is similar to that of the float. Although detailed engineering considerations are beyond the scope of this study, it is believed that this can be achieved, by spreading the reference body of water across several PTO tubes, for example. The w2Gr range was chosen so that the point mass is always below the centre of gravity of the float and therefore does not reduce its stability. It can be noticed that the range of w2Gr starts at −0.05 m, which is within the float and not below it. From an engineering point of view, this would correspond to having the PTO tube integrated to the float. The w2Gr lower limit was set to −0.5×cylinder draft as too large a distance between the float and the tube could be impractical from an engineering perspective.The parameters investigated and their range are given by CW) which corresponds to the ratio of the energy harvested by the WEC over the incoming wave energy available in a wave front whose width is equal to that of the device. More information on that concept can be found in  range. This approach makes it possible to control and investigate the influence of each metric on the overall score. Three weight combinations are defined as part of the optimization process:(iii)m2,w2Gr,θ0 and α) are imported into the statistical analysis package R  Nsm−1 range, and the variability seen in It can be observed that the optimal 20 would be expected to have a higher score20 than the two other solutions but that is not the case. As the three solutions are very close, these discrepancies are thought to be due to noise in the optimization process. These discrepancies would be less likely to occur if the two optimization metrics did not lead to similar results as is the case here.scores investigated. As mentioned earlier the parameter values for the three scores are quite similar and hence so are the curves. The curves exhibit a ‘double peak’. From a hydrodynamics point of view, the first peak corresponds to the system resonance in pitch and the second one to the resonance in heave. A clear advantage of the sloped PTO configuration is to be able to harness energy from both pitch/surge and heave motions. Moreover, the capture width ratio curve only dips slightly between the two peaks, providing the device with a wide bandwidth of high capture with ratio (above 0.7). This feature is advantageous in that it allows the system to perform efficiently over a broad range of sea states without the need for complex control. In comparison, a purely heaving system has a narrower bandwidth.5.m2 is the maximum of the range considered, i.e. m2=m1. For w2Gr it is the lowest value of the range: w2Gr=−0.25 m. For the PTO angle and damping, the optimum values vary slightly with the score chosen. It is respectively between 293 and 327 Nms−1 and around 50°.The concept of inclining the PTO axis of a free-floating WEC reacting against water inertia has been investigated using numerical modelling. The shape of the WEC has deliberately been kept to that of a simple vertical axis circular cylinder and the study has focused on PTO parameters. The derivation of the numerical modelling has been presented in detail. The thorough validation of that model has been carried out by ensuring that the results made physical sense and by comparing these with benchmark numerical data. This successful validation has built the necessary confidence into the model so that it could reliably be used for the parametric study and the optimization which has investigated the impact of the mass of the point mass, of its vertical position, of the angle of the PTO damper and of the damping value on the WEC performance. A multi-linear regression method has been used to find the set of parameters which provides the best performance in terms of linear combinations of capture width area and mean capture width period. Four thousand WEC configurations have been generated in that process. The optimum values for Given the simplicity of the WEC shape and the fact that the numerical modelling technique used is frequency domain BEM, computations for each configuration was quick. This makes it possible to compute the performance of a large number of configurations. In that context, it would have been possible to identify a best performing configuration by carrying out a systematic exploration of the parametric domain with a fine parametric resolution. It should, however, be noted that the fitting of the analytical model and the subsequent optimization did yield some performance improvement over the best of the 4000 initial cases, which is already a large number of configurations. But perhaps more importantly, the optimization approach used here has provided an understanding of the impact of each parameter on the device performance and behaviour and this could not have been achieved with a systematic ‘brute-force’ optimization.The parametric study shows, in a comprehensive manner, the clear advantage of an inclined PTO direction versus a vertical one. This is physically due to the fact that inclined configurations exploit resonance in both heave and pitch, thus widening the period range over which high capture width ratios are observed. These fundamental results pave the way for exploring further WEC concepts featuring sloped PTOs.